Formulations of antibody molecules to dengue virus

ABSTRACT

This disclosure relates to formulations of peptide agents, e.g., antibodies and antigen-binding fragments thereof, that bind dengue viruses, and methods of their use.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/313,558, filed Mar. 25, 2016. The contents of the aforementionedapplication are hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 20, 2017, isnamed P2029-701010_SL.txt and is 72,904 bytes in size.

BACKGROUND

Dengue virus is a positive-sense RNA virus belonging to the Flavivirusgenus of the family Flaviviridae. Dengue virus is widely distributedthroughout the tropical and semitropical regions of the world and istransmitted to humans by mosquito vectors. Dengue virus is a leadingcause of hospitalization and death in children in at least eighttropical Asian countries (WHO, 1997. Dengue haemorrhagic fever:diagnosis, treatment prevention and control—2nd ed. Geneva: WHO). Thereare four serotypes of dengue virus (DV-1, DV-2, DV-3, and DV-4) whichannually cause an estimated 50-100 million cases of dengue fever and500,000 cases of the more severe form of dengue virus infection, denguehemorrhagic fever/dengue shock syndrome (DHF/DSS) (Gubler, D. J. &Meltzer, M. 1999 Adv Virus Res 53:35-70). DHF/DSS is seen predominatelyin children and adults experiencing a second dengue virus infection witha serotype different than that of their first dengue virus infection andin primary infection of infants who still have circulatingdengue-specific maternal antibody (Burke, D. S. et al. 1988 Am J TropMed Hyg 38:172-80; Halstead, S. B. et al. 1969 Am J Trop Med Hyg18:997-1021; Thein, S. et al. 1997 Am J Trop Med Hyg 56:566-72).

The different serotypes of dengue virus differ at the amino acid levelby about 25-40% and have antigenic differences, and this variation hashindered efforts to produce a therapy effective against all serotypes.

All four dengue virus serotypes display an E (envelope) protein on theviral surface. The E protein contributes to the attachment of the virusto a host cell. The E protein comprises a DI domain (a nine-strandedbeta-barrel) a DII domain (a domain implicated in fusion with the hostcell), and a DIII domain (an immunoglobulin-like domain). The humoralresponse to E protein in humans generally targets the DI and DIIregions, with much of the antibodies exhibiting high cross-serotypereactivity but low neutralization activity.

There is a need in the art for new prophylactic and therapeutictreatments for dengue virus, and especially for treatments that areeffective against all four serotypes of the virus.

SUMMARY

This disclosure provides, at least in part, formulations of antibodymolecules that bind to the dengue virus, for example, the dengue virus Eprotein, and which comprise functional and structural propertiesdisclosed herein. In some embodiments, the antibody molecules bind tothe “A” beta-strand of EDIII (the E protein DIII domain). In someembodiments, the antibody molecules bind to and/or neutralize at least1, 2, 3, or 4 dengue virus serotypes, e.g., DV-1, DV-2, DV-3, and DV-4.In some embodiments, the antibody molecule is selected from Table 1. Insome embodiments, the antibody molecules comprise a deletion of VH S26and/or a VH T33V substitution compared to antibody A11. These mutations,in some embodiments, may improve one or more properties, e.g., improveantibody affinity for one or more dengue virus serotypes, for exampleserotype DV-4. In some embodiments, the antibody molecule targets a siteon EDIII that is conserved across all four dengue serotypes. Nucleicacid molecules encoding the antibody molecules, expression vectors, hostcells, pharmaceutical compositions, and methods for making the antibodymolecules are also provided. The anti-dengue antibody moleculesdisclosed herein can be used (alone or in combination with other agentsor therapeutic modalities) to treat, prevent and/or diagnose denguevirus, e.g., DV-1, DV-2, DV-3, or DV-4.

In an aspect, the disclosure features a formulation, e.g., apharmaceutical formulation or composition, comprising an anti-dengueantibody molecule described herein, e.g., D88, and a buffering agent.

In an embodiment, the antibody molecule is present at a concentration ofabout 5 mg/mL to about 150 mg/mL, e.g., about 10 mg/mL to about 100mg/mL, about 10 mg/mL to about 50 mg/mL, about 10 mg/mL to about 40mg/mL, about 15 mg/mL to about 75 mg/mL, about 20 mg/mL to about 50mg/mL, about 20 mg/mL to about 30 mg/mL, about 15 mg/mL to about 25mg/mL, about 25 mg/mL to about 35 mg/mL, about 5 mg/mL to about 50mg/mL, about 50 mg/mL to about 100 mg/mL, about 100 mg/mL to about 150mg/mL, about 5 mg/mL to about 25 mg/mL, about 10 mg/mL to about 30mg/mL, about 20 mg/mL to about 40 mg/mL, about 30 mg/mL to about 50mg/mL, about 40 mg/mL to about 60 mg/mL, about 50 mg/mL to about 70mg/mL, about 60 mg/mL to about 80 mg/mL, about 70 mg/mL to about 90mg/mL, about 80 mg/mL to about 100 mg/mL, about 90 mg/mL to about 110mg/mL, or about 100 mg/mL to about 120 mg/mL, e.g., about 150 mg/mL orless, about 100 mg/mL or less, about 50 mg/mL or less, about 25 mg/mL orless, or about 10 mg/mL or less, e.g., about 5 mg/mL, about 10 mg/mL,about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 60 mg/mL,about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, or about150 mg/mL.

In an embodiment, the antibody molecule is present at a concentration ofabout 10 mg/mL to about 50 mg/mL, e.g., about 10 mg/mL to about 40mg/mL, e.g., about 20 mg/mL to about 30 mg/mL, e.g., about 25 mg/mL. Inan embodiment, the antibody molecule is present at a concentration ofabout 25 mg/mL.

In an embodiment, the antibody molecule comprises a heavy chain (HC)immunoglobulin variable region segment comprising:

an HC CDR1 comprising the sequence DVYMS (SEQ ID NO: 3) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom, providedthat V is unchanged),

an HC CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4) (ora sequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom, optionally provided that L is unchanged), and

an HC CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom).

In another embodiment, the antibody molecule comprises a light chainimmunoglobulin (LC) variable region segment comprising:

an LC CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom),

an LC CDR2 comprising the sequence RASELQW (SEQ ID NO: 7) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom), and

an LC CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8) (or asequence that differs by no more than, 1, 2, or 3 amino acidstherefrom).

In an embodiment, the antibody molecule comprises:

(a) a heavy chain (HC) immunoglobulin variable region segmentcomprising:

an HC CDR1 comprising the sequence DVYMS (SEQ ID NO: 3) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom, providedthat V is unchanged),

an HC CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4) (ora sequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom, optionally provided that L is unchanged), and

an HC CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom); and

(b) a light chain (LC) immunoglobulin variable region segmentcomprising:

an LC CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom),

an LC CDR2 comprising the sequence RASELQW (SEQ ID NO: 7) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom), and

an LC CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8) (or asequence that differs by no more than, 1, 2, or 3 amino acidstherefrom).

In an embodiment, the antibody molecule comprises:

(a) a heavy chain (HC) immunoglobulin variable region segmentcomprising:

an HC CDR1 comprising the sequence DVYMS (SEQ ID NO: 3),

an HC CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4), and

an HC CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5); and

(b) a light chain (LC) immunoglobulin variable region segmentcomprising:

an LC CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6),

an LC CDR2 comprising the sequence RASELQW (SEQ ID NO: 7), and

an LC CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8).

In an embodiment, the antibody molecule comprises the heavy chainvariable region (VH) amino acid sequence of SEQ ID NO: 1. In anotherembodiment, the antibody molecule comprises the light chain variableregion (VL) amino acid sequence of SEQ ID NO: 2. In yet anotherembodiment, the antibody molecule comprises the VH amino acid sequenceof SEQ ID NO: 1 and the VL amino acid sequence of SEQ ID NO: 2.

In an embodiment, the buffering agent is present at a concentration ofabout 5 mM to about 150 mM, e.g., about 10 mM to about 100 mM, about 20mM to about 50 mM, about 1 mM to about 50 mM, about 5 mM to about 20 mM,about 10 mM to about 40 mM, about 5 mM to about 30 mM, about 10 mM toabout 25 mM, e.g., about 150 mM or less, about 100 mM or less, about 75mM or less, about 50 mM or less, about 40 mM or less, about 30 mM orless, about 25 mM or less, or about 10 mM or less, e.g., about 5 mM,about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM,about 110 mM, about 120 mM, about 130 mM, about 140 mM, or about 150 mM.

In an embodiment, the buffering agent is present at a concentration ofabout 10 mM to about 50 mM, e.g., about 20 to about 40 mM, e.g., about25 mM. In an embodiment, the buffering agent is present at aconcentration of about 25 mM. In another embodiment, the buffering agentis present at a concentration of about 1 mM to about 50 mM, e.g., about5 to about 20 mM, e.g., about 10 mM. In an embodiment, the bufferingagent is present at a concentration of about 10 mM.

In an embodiment, the buffering agent is a citrate buffer or a phosphatebuffer. In an embodiment, the buffering agent comprises sodiumphosphate. In one embodiment, the buffering agent comprises sodiumphosphate at a concentration of about 5 mM to about 150 mM, e.g., about10 mM to about 50 mM, e.g., about 25 mM. In another embodiment, thebuffering agent comprises sodium citrate. In one embodiment, thebuffering agent comprises sodium citrate at a concentration of about 5mM to about 150 mM, e.g., about 5 mM to about 50 mM, about 10 mM toabout 30 mM, e.g., about 10 mM or about 25 mM.

In another embodiment, the buffering agent is a histidine buffer. In oneembodiment, the buffering agent comprises histidine at a concentrationof about 5 mM to about 150 mM, e.g., about 10 mM to about 50 mM or about20 mM to about 40 mM, e.g., about 25 mM.

In an embodiment, the buffering agent provides a pH of about 5.5 toabout 7.5, e.g., about 6.0 to about 7.0, e.g., about 6.3 to about 6.7 orabout 6.4 to about 6.6, e.g., about 5.5, about 6, about 6.5, or about 7.In one embodiment, the buffering agent comprises sodium phosphate andprovides a pH of about 7.0. In another embodiment, buffering agentcomprises sodium citrate and provides a pH of about 6.0 or 6.5. In afurther embodiment, the buffering agent comprises histidine and providesa pH of about 6.5. In another embodiment, the buffering agent comprisesarginine and provides a pH of about 7.0.

In an embodiment, the formulation further comprises a tonicity agent.

In an embodiment, the tonicity agent is present at a concentration ofabout 10 mM to about 500 mM, about 50 mM to about 200 mM, e.g., about 60mM to about 190 mM, about 70 mM to about 180 mM, about 80 mM to about170 mM, about 90 mM to about 160 mM, about 100 mM to about 150 mM, about145 mM to about 155 mM, about 140 mM to about 160 mM, about 135 mM toabout 165 mM, about 130 mM to about 170 mM, about 120 mM to about 180mM, about 70 mM to about 130 mM, about 80 to about 120 mM, about 90 toabout 110 mM, about 110 mM to about 190 mM, about 100 mM to about 200mM, about 50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mMto about 80 mM, about 100 mM to about 150 mM, or about 120 to about 150mM, e.g., about 200 mM or less, about 160 mM or less about 150 mM orless, about 120 mM or less, about 110 mM or less, about 100 mM or less,about 80 or less, or about 75 mM or less, e.g., about 50 mM, about 60mM, about 70 mM, about 75 mM, about 80 mM, about 90 mM, about 100 mM,about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM,about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM.

In an embodiment, the tonicity agent is used at a concentration of about50 mM to about 200 mM, about 75 mM to about 150 mM, about 120 mM toabout 180 mM, e.g., about 140 mM to about 160 mM, e.g., about 150 mM. Inanother embodiment, the tonicity agent is used at a concentration ofabout 50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM toabout 80 mM, e.g., about 75 mM. In another embodiment, the tonicityagent is used at a concentration of about 50 mM to about 200 mM, about60 mM to about 130 mM, about 70 mM to about 120 mM, about 80 mM to about110 mM, e.g., about 100 mM.

In an embodiment, the tonicity agent comprises sodium chloride. In anembodiment, the tonicity agent comprises sodium chloride and is used ata concentration of about 140 mM to about 160 mM, e.g., about 150 mM. Inan embodiment, the tonicity agent comprises sodium chloride and is usedat a concentration of about 55 mM to about 95 mM, e.g., about 75 mM. Inan embodiment, the tonicity agent comprises sodium chloride and is usedat a concentration of about 80 mM to about 120 mM, e.g., about 100 mM.

In an embodiment, the tonicity agent provides a tonicity of about 150mOsm/L to about 400 mOsm/L, about 160 mOsm/L to about 390 mOsm/L, about170 mOsm/L to about 380 mOsm/L, about 180 mOsm/L to about 370 mOsm/L,about 190 mOsm/L to about 360 mOsm/L, about 200 mOsm/L to about 370mOsm/L, about 210 mOsm/L to about 360 mOsm/L, about 220 mOsm/L to about350 mOsm/L, about 200 mOsm/L to about 350 mOsm/L, about 220 mOsm/L toabout 340 mOsm/L, or about 220 mOsm/L to about 340 mOsm/L, about 230mOsm/L to about 330 mOsm/L, or about 240 mOsm/L to about 320 mOsm/L, orabout 250 mOsm/L to about 300 mOsm/L, e.g., about 250 mOsm/L, about 260mOsm/L, about 270 mOsm/L, about 280 mOsm/L, about 290 mOsm/L, about 300mOsm/L, about 310 mOsm/L, about 320 mOsm/L, about 330 mOsm/L, about 340mOsm/L, or about 350 mOsm/L.

In an embodiment, the formulation does not comprise a tonicity agent.

In an embodiment, the formulation has a pH of about 5.5 to about 7,e.g., about 6 to about 6.5, e.g., about 5.5, about 6, about 6.5, orabout 7.

In an embodiment, the formulation further comprises a surfactant, e.g.,a nonionic surfactant.

In an embodiment, the surfactant is present at a concentration of about0.005% to about 0.1% (w/v), e.g., about 0.01% to about 0.05%, about0.015% to about 0.04%, about 0.02% to about 0.03%, about 0.01% to about0.03%, about 0.02% to about 0.04%, about 0.01% to about 0.02%, about0.02% to about 0.1%, about 0.005% to about 0.05%, or about 0.05% toabout 0.1%, e.g., about 0.1% or less, about 0.075% or less, about 0.05%or less, about 0.02% or less, or about 0.01% or less, e.g., about0.005%, about 0.01%, about 0.015%, about 0.02%, about 0.025%, about0.03%, about 0.035%, about 0.04%, about 0.05%, about 0.06%, about 0.07%,about 0.08%, about 0.09%, or about 0.1%.

In an embodiment, the surfactant is present at a concentration of about0.01% to about 0.05%, e.g., about 0.02%.

In an embodiment, the surfactant is polysorbate 80 (TWEEN® 80). In anembodiment, the surfactant is polysorbate 80 and is present atconcentration of about 0.01% and about 0.05%, e.g., about 0.01% to about0.03%, e.g., about 0.02%.

In an embodiment, the formulation does not comprise a surfactant, e.g.,a nonionic surfactant.

In an embodiment, the formulation further comprises a stabilizing agent.

In an embodiment, the stabilizing agent is present at a concentration ofabout 50 mM to about 200 mM, e.g., about 50 mM to about 150 mM, about 60mM to about 190 mM, about 70 mM to about 180 mM, about 75 mM to about125 mM, or about 75 mM to about 100 mM, about 80 mM to about 170 mM,about 90 mM to about 160 mM, about 100 mM to about 150 mM, about 145 mMto about 155 mM, about 140 mM to about 160 mM, about 135 mM to about 165mM, about 130 mM to about 170 mM, about 120 mM to about 180 mM, about 70mM to about 130 mM, about 80 to about 120 mM, about 90 to about 110 mM,about 110 mM to about 190 mM, about 100 mM to about 200 mM, about 50 mMto about 100 mM, about 60 mM to about 90 mM, about 70 mM to about 80 mM,about 100 mM to about 150 mM, or about 150 mM to about 120 mM, e.g.,about 200 mM or less, about 160 mM or less about 150 mM or less, about120 mM or less, about 110 mM or less, about 100 mM or less, about 80 orless, or about 75 mM or less, e.g., about 50 mM, about 60 mM, about 70mM, about 75 mM about 80 mM, about 90 mM, about 100 mM, about 110 mM,about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM,about 170 mM, about 180 mM, about 190 mM, or about 200 mM.

In an embodiment, the stabilizing agent is used at a concentration ofabout 50 mM to about 200 mM, about 75 mM to about 150 mM, about 120 mMto about 180 mM, e.g., about 140 mM to about 160 mM, e.g., about 150 mM.In another embodiment, the stabilizing agent is used at a concentrationof about 50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mMto about 80 mM, e.g., about 75 mM.

In an embodiment, the stabilizing agent is an amino acid. In anembodiment, the amino acid is glycine, histidine, arginine, methionine,proline, lysine, glutamic acid, or a combination thereof.

In an embodiment, the stabilizing agent comprises arginine. In anembodiment, the stabilizing agent comprises arginine and is used at aconcentration of about 50 mM to about 200 mM or about 75 mM to about 150mM. In an embodiment, the stabilizing agent comprises arginine and isused at a concentration of about 140 mM to about 160 mM, e.g., about 150mM. In an embodiment, the stabilizing agent comprises arginine and isused at a concentration of about 55 mM to about 95 mM, e.g., about 75mM. In an embodiment, the formulation comprises histidine, e.g., as astabilizing agent. In an embodiment, the formulation comprises about 10mM to about 40 mM, e.g., about 25 mM, histidine.

In an embodiment, the formulation comprises:

(a) an antibody molecule described herein at a concentration of about 10to about 50 mg/mL, e.g., about 10 to about 40 mg/mL, e.g., about 20 toabout 30 mg/mL, e.g., about 25 mg/mL;

(b) a buffering agent, e.g., histidine, at a concentration of about 10mM to about 50 mM, e.g., about 20 mM to about 40 mM, e.g., about 25 mM;

(c) a tonicity agent, e.g., sodium chloride, at a concentration of about50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM to about80 mM, e.g., about 75 mM; and

(d) a stabilizing agent, e.g., arginine, at a concentration of about 50mM to about 200 mM, e.g., about 60 mM to about 100 mM, e.g., about 75mM;

wherein the pH of the formulation is about 5.5 to about 7.0, e.g., about6.5.

In an embodiment, the formulation comprises about 25 mg/mL of anantibody molecule described herein, about 25 mM histidine, about 75 mMsodium chloride, and about 75 mM arginine, wherein the pH of theformulation is about 6.5.

In an embodiment, the formulation comprises:

(a) an antibody molecule described herein at a concentration of about 10to about 50 mg/mL, e.g., about 10 to about 40 mg/mL, e.g., about 20 toabout 30 mg/mL, e.g., about 25 mg/mL;

(b) a buffering agent, e.g., histidine, at a concentration of about 10mM to about 50 mM, e.g., about 20 mM to about 40 mM, e.g., about 25 mM;

(c) a tonicity agent, e.g., sodium chloride, at a concentration of about50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM to about80 mM, e.g., about 75 mM;

(d) a stabilizing agent, e.g., arginine, at a concentration of about 50mM to about 200 mM, e.g., about 60 mM to about 100 mM, e.g., about 75mM; and

(e) a surfactant, e.g., polysorbate 80, at a concentration of about0.01% to about 0.04%, e.g., about 0.02%;

wherein the pH of the formulation is about 5.5 to about 7.0, e.g., about6.5.

In some embodiments, the formulation comprises about 25 mg/mL of anantibody molecule described herein, about 25 mM histidine, about 75 mMsodium chloride, about 75 mM arginine, and 0.02% polysorbate 80, whereinthe pH of the pharmaceutical composition is about 6.5.

In an embodiment, the formulation comprises a carbohydrate, e.g., apolyol or a sugar. In an embodiment, the carbohydrate is sucrose,trehalose, mannitol, dextran, sorbitol, inositol, glucose, fructose,lactose, xylose, mannose, maltose, raffinose, or a combination thereof.

In some embodiments, the carbohydrate is sucrose. In some embodiments,sucrose is present at a concentration of about 50 mM to about 150, e.g.,about 60 mM to about 140 mM, about 70 mM to about 130 mM, about 80 mM toabout 120 mM, about 90 mM to about 110 mM, about 75 mM to about 100 mM,about 75 mM to about 125 mM, about 50 mM to about 100 mM, or about 100mM to about 150 mM, about 50 mM to about 90 mM, about 60 mM to about 80mM, e.g., about 200 mM or less, about 190 mM or less, about 180 mM orless, about 170 mM or less, about 160 mM or less, about 150 mM or less,about 140 mM or less, about 130 mM or less, about 120 mM or less, about110 mM or less, about 100 mM or less, about 90 mM or less, about 80 mMor less, about 75 mM or less, or about 60 mM or less, e.g., about 200mM, about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 150mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100mM, about 90 mM, about 80 mM, about 75 mM, or about 60 mM. In someembodiments, sucrose is present at a concentration of about 75 mM. Insome embodiments, sucrose is present at a concentration of about 100 mM.In some embodiments, sucrose is present at a concentration of about 150mM.

In some embodiments, the carbohydrate is sorbitol. In some embodiments,sorbitol is present at a concentration of about 50 mM to about 150 mM,e.g., about 70 mM to about 140 mM, about 90 mM to about 130 mM, e.g.,about 150 mM or less, about 145 mM or less, about 140 mM or less, about135 mM or less, about 130 mM or less, about 125 mM or less, about 120 mMor less, about 115 mM or less, about 110 mM or less, about 105 mM orless, or about 100 mM or less, e.g., about 150 mM, about 145 mM, about140 mM, about 135 mM, about 130 mM, about 125 mM, about 120 mM, about115 mM, about 110 mM, about 105 mM, or about 100 mM. In someembodiments, sorbitol is present at a concentration of about 125 mM.

In an embodiment, the formulation comprises:

(a) an antibody molecule described herein at a concentration of about 10to about 50 mg/mL, e.g., about 10 to about 40 mg/mL, e.g., about 20 toabout 30 mg/mL, e.g., about 25 mg/mL;

(b) a buffering agent, e.g., histidine, at a concentration of about 10mM to about 50 mM, e.g., about 20 mM to about 40 mM, e.g., about 25 mM;

(c) a tonicity agent, e.g., sodium chloride, at a concentration of about50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM to about80 mM, e.g., about 75 mM; and

(d) a carbohydrate, e.g., sucrose, at a concentration of about 50 mM toabout 150 mM, e.g., about 90 mM to about 110 mM, e.g., about 100 mM;

wherein the pH of the formulation is about 5.5 to about 7.0, e.g., about6.5.

In an embodiment, the formulation comprises about 25 mg/mL of antibodymolecule described herein, about 25 mM histidine, about 75 mM sodiumchloride, and about 100 mM sucrose, wherein the pH of the formulation isabout 6.5.

In an embodiment, the formulation comprises:

(a) an antibody molecule described herein at a concentration of about 10to about 50 mg/mL, e.g., about 10 to about 40 mg/mL, e.g., about 20 toabout 30 mg/mL, e.g., about 25 mg/mL;

(b) a buffering agent, e.g., histidine, at a concentration of about 10mM to about 50 mM, e.g., about 20 mM to about 40 mM, e.g., about 25 mM;

(c) a tonicity agent, e.g., sodium chloride, at a concentration of about50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM to about80 mM, e.g., about 75 mM;

(d) a carbohydrate, e.g., sucrose, at a concentration of about 50 mM toabout 150 mM, e.g., about 90 mM to about 110 mM, e.g., about 100 mM; and

(e) a surfactant, e.g., polysorbate 80, at a concentration of about0.01% to about 0.04%, e.g., about 0.02%;

wherein the pH of the formulation is about 5.5 to about 7.0, e.g., about6.5.

In an embodiment, the formulation comprises about 25 mg/mL of anantibody molecule described herein, about 25 mM histidine, about 75 mMsodium chloride, about 100 mM sucrose, and about 0.02% polysorbate 80,wherein the pH of the formulation is about 6.5.

In an embodiment, the formulation comprises:

(a) an antibody molecule described herein at a concentration of about 10to about 50 mg/mL, e.g., about 10 to about 40 mg/mL, e.g., about 20 toabout 30 mg/mL, e.g., about 25 mg/mL;

(b) a buffering agent, e.g., sodium citrate, at a concentration of about15 mM to about 50 mM, e.g., about 25 mM;

(c) a tonicity agent, e.g., sodium chloride, at a concentration of about50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM to about80 mM, e.g., about 75 mM; and

(d) a carbohydrate, e.g., sucrose, at a concentration of about 50 mM toabout 150 mM, e.g., about 60 mM to about 100 mM, e.g., about 75 mM;

wherein the pH of the formulation is about 5.5 to about 7.0, e.g., about6.5.

In an embodiment, the formulation comprises about 25 mg/mL of anantibody molecule described herein, about 25 mM sodium citrate, about 75mM sodium chloride, and about 75 mM sucrose, wherein the pH of theformulation is about 6.5.

In an embodiment, the formulation further comprises a polymer, e.g., ahydrophilic polymer. In an embodiment, the polymer is a polyethyleneglycol (PEG), dextran, hydroxyl ethyl starch (HETA), or gelatin.

In an embodiment, the formulation further comprises a preservative. Inan embodiment, the preservative is benzyl alcohol, m-cresol, or phenol.

In an embodiment, the level of high molecular weight (HMW) species inthe formulation is less than about 20%, less than about 19%, less thanabout 18%, less than about 17%, less than about 16%, less than about15%, less than about 14%, less than about 13%, less than about 12%, lessthan about 10%, less than about 9%, less than about 8%, less than about7%, less than about 6%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2%, or less than 1%, e.g., beforestorage, or after storage for at least about 1 week at 4° C., or afterstorage for at least about 1 week at 5° C., or after storage for atleast about 1 week at 40° C., at least about 1 week at 45° C., at leastabout 2 weeks at 4° C., at least about 2 weeks at 5° C., at least about2 weeks at 40° C., at least about 2 weeks at 45° C., at least about 3weeks at 4° C., at least about 3 weeks at 5° C., at least about 3 weeksat 40° C., at least about 3 weeks at 45° C., at least about 4 weeks at4° C., at least about 4 weeks at 5° C., at least about 4 weeks at 40°C., at least about 4 weeks at 45° C., at least about 5 weeks at 4° C.,at least about 5 weeks at 5° C., at least about 5 weeks at 40° C., atleast about 5 weeks at 45° C., at least about 6 weeks at 4° C., at leastabout 6 weeks at 5° C., at least about 6 weeks at 40° C., at least about6 weeks at 45° C. In an embodiment, the level of HMW species is lessthan about 2% before storage. In an embodiment, the level of HMW speciesis less than about 2% after storage for 4 weeks at 5° C.

In an embodiment, the level of low molecular weight (LMW) species in theformulation is less than about 20%, less than about 19%, less than about18%, less than about 17%, less than about 16%, less than about 15%, lessthan about 14%, less than about 13%, less than about 12%, less thanabout 10%, less than about 9%, less than about 8%, less than about 7%,less than about 6%, less than about 5%, less than about 4%, less thanabout 3%, less than about 2%, or less than 1%, e.g., before storage orafter storage for at least about 1 week at 4° C., or after storage forat least about 1 week at 5° C., or after storage for at least about 1week at 40° C., at least about 1 week at 45° C., at least about 2 weeksat 4° C., at least about 2 weeks at 5° C., at least about 2 weeks at 40°C., at least about 2 weeks at 45° C., at least about 3 weeks at 4° C.,at least about 3 weeks at 5° C., at least about 3 weeks at 40° C., atleast about 3 weeks at 45° C., at least about 4 weeks at 4° C., at leastabout 4 weeks at 5° C., at least about 4 weeks at 40° C., at least about4 weeks at 45° C., at least about 5 weeks at 4° C., at least about 5weeks at 5° C., at least about 5 weeks at 40° C., at least about 5 weeksat 45° C., at least about 6 weeks at 4° C., at least about 6 weeks at 5°C., at least about 6 weeks at 40° C., at least about 6 weeks at 45° C.In an embodiment, the level of LMW species is less than about 1% beforestorage.

In an embodiment, the level of HMW and LMW species in the formulation isless than about 30%, less than about 29%, less than about 28%, less thanabout 27%, less than about 26%, less than about 25%, less than about24%, less than about 23%, less than about 22%, less than about 21%, lessthan about 20%, less than about 19%, less than about 18%, less thanabout 17%, less than about 16%, less than about 15%, less than about14%, less than about 13%, less than about 12%, less than about 10%, lessthan about 9%, less than about 8%, less than about 7%, less than about6%, less than about 5%, less than about 4%, less than about 3%, lessthan about 2%, or less than 1%, e.g., before storage or after storagefor at least about 1 week at 4° C., or after storage for at least about1 week at 5° C., or after storage for at least about 1 week at 40° C.,at least about 1 week at 45° C., at least about 2 weeks at 4° C., atleast about 2 weeks at 5° C., at least about 2 weeks at 40° C., at leastabout 2 weeks at 45° C., at least about 3 weeks at 4° C., at least about3 weeks at 5° C., at least about 3 weeks at 40° C., at least about 3weeks at 45° C., at least about 4 weeks at 4° C., at least about 4 weeksat 5° C., at least about 4 weeks at 40° C., at least about 4 weeks at45° C., at least about 5 weeks at 4° C., at least about 5 weeks at 5°C., at least about 5 weeks at 40° C., at least about 5 weeks at 45° C.,at least about 6 weeks at 4° C., at least about 6 weeks at 5° C., atleast about 6 weeks at 40° C., at least about 6 weeks at 45° C. In anembodiment, the level of HMW and LMW species is less than about 2%before storage.

In an embodiment, about 90% or more, about 92% or more, about 94% ormore, about 95% or more, about 96% or more, about 97% or more, about 98%or more, or about 99% or more of the antibody molecules in theformulation are present as monomers, e.g., before storage, or afterstorage for at least about 1 week at 5° C., at least about 1 week at 40°C., at least about 2 weeks at 5° C., at least about 2 weeks at 40° C.,at least about 3 weeks at 5° C., at least about 3 weeks at 40° C., atleast about 4 weeks at 5° C., at least about 4 weeks at 40° C., at leastabout 5 weeks at 5° C., at least about 5 weeks at 40° C., at least about6 weeks at 5° C., or at least about 6 weeks at 40° C. In an embodiment,about 98% or more of the antibody molecules in the formulation arepresent as monomers before storage. In an embodiment, about 98% or moreof the antibody molecules in the formulation are present as monomersafter storage for 4 weeks at 5° C. In an embodiment, about 95% or moreof the antibody molecules in the formulation are present as monomersafter storage for 4 weeks at 40° C.

In an embodiment, the level of monomers species is determined by highperformance liquid chromatography (HPLC), e.g., gel permeation highperformance liquid chromatography (GP HPLC). In another embodiment, themonomeric nature of the antibody molecule is determined by sizeexclusion-high performance liquid chromatography (SEC-HPLC), a bindingassay, a surface charge assay, a bioassay, or the ratio of HMW speciesto LMW species. In another embodiment, the level of HMW species isdetermined by dynamic light scattering.

In an embodiment, the purity of the antibody molecule in theformulation, e.g., after storage for 4 weeks at 5° C., is at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%. In an embodiment, thepurity of the antibody molecule in the formulation, e.g., after storagefor 4 weeks at 40° C., is at least about 80%, least about 82%, leastabout 84%, least about 86%, least about 88%, least about 90%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%. In an embodiment, the purity (or heterogeneity) of theantibody molecule is determined by detecting the intact heavy and lightchains (e.g., in a reduced sample) or intact immunoglobulins (e.g., in anon-reduced sample) in the formulation.

In an embodiment, the formulation is a liquid formulation. In anotherembodiment, the formulation is a lyophilized formulation.

In an embodiment, the formulation is for use in treating or preventingdengue virus in a subject. In an embodiment, the formulation is for usein treating a subject having dengue virus. In another embodiment, theformulation is for use in preventing a subject from having dengue virus.

In another aspect, the disclosure features a device, e.g., an injectiondevice, comprising a formulation described herein, e.g., apharmaceutical formulation described herein.

In another aspect, the disclosure features a container, comprising aformulation described herein, e.g., a pharmaceutical formulationdescribed herein.

In yet another aspect, the disclosure features a kit, comprising aformulation described herein, or one or more containers comprising aformulation described herein, e.g., a pharmaceutical formulationdescribed herein, and instructions for administration of the formulationto a subject.

In another aspect, the disclosure features a method of making aformulation described herein, the method comprising combining anantibody molecule described herein with one or more (e.g., two, three,four, five, or more) components (e.g., a buffering agent, a tonicityagent, a stabilizing agent, a surfactant, a carbohydrate, or an aminoacid, as described herein) of a formulation described herein, e.g., apharmaceutical formulation described herein.

In still another aspect, the disclosure features a method of treating orpreventing dengue virus, the method comprising administering to asubject having dengue virus, or at risk of having dengue virus, aneffective amount of a formulation described herein, e.g., apharmaceutical formulation described herein, thereby treating orpreventing dengue virus.

In an aspect, the disclosure features a formulation described herein,e.g., a pharmaceutical formulation described herein, for use in treatingor preventing dengue virus.

In another aspect, the disclosure features use of a formulationdescribed herein, e.g., a pharmaceutical formulation described herein,in the manufacture of a medicament for the treatment or prevention ofdengue virus.

Any of the formulations and uses disclosed herein can include anantibody molecule to dengue virus, e.g., an anti-dengue antibodymolecule described herein. In some embodiments, the antibody molecule(e.g., an isolated, recombinant, or humanized antibody molecule) has oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, or all) of the following properties fromList 1:

-   -   a) Binds to EDIII (e.g., one or more EDIII from any dengue virus        serotype, e.g., from DV-1, DV-2, DV-3, or DV-4, e.g., all four        EDIII from DV-1, DV-2, DV-3, or DV-4) with high affinity, e.g.,        with a dissociation constant (K_(D)) of less than about 100 nM,        typically about 10 nM, and more typically, about 10-0.01 nM,        about 5-0.01 nM, about 3-0.05 nM, about 1-0.1 nM, or stronger,        e.g., less than about 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4,        3, 2, 1, 0.5, 0.2, 0.1, 0.05, or 0.01 nM,    -   b) Binds to DV-4 EDIII with high affinity, e.g., with a        dissociation constant (K_(D)) of less than about 100 nM, e.g.,        about 10 nM, e.g., about 10-1 nM or stronger, e.g., less than        about 10, 8, 6, 5, 4, or 3 nM,    -   c) Binds to DV-4 and/or DV-3 EDIII domain with a greater        affinity than antibody A11 (also referred to as 4E5A herein)        and/or antibody 4E11, e.g., at least 2, 3, 4, 5, 6, 8, 10, 12,        15, 100, 1,000, 5,000, or 10,000-fold greater affinity,    -   d) Neutralizes dengue virus (e.g., one or more of DV-1, DV-2,        DV-3, and DV-4, e.g., all of DV-1, DV-2, DV-3, and DV-4), e.g.,        in a focus reduction neutralization test or a related test for        evaluating neutralization of viral activity,    -   e) Neutralizes DV-4 with an improved IC50 compared to antibody        A11 and/or antibody 4E11, e.g., at least 2, 3, 4, 5, 6, 8, 10,        12, 25, 50, 75, 100, or 1,000-fold improved IC50, e.g., in a        focus reduction neutralization test or a related test for        evaluating neutralization of viral activity,    -   f) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution) at        one or more positions relative to A11, e.g., in the VH and/or        VL, e.g., in one or more CDRs or framework regions,    -   g) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a substitution, e.g., a T33V substitution, in the heavy        chain CDR1 region relative to A11,    -   h) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a deletion, at position 26 in the heavy chain FW1 relative        to A11,    -   i) Has both a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a substitution, e.g., a T33V mutation in the heavy chain        CDR1 region relative to A11 and a mutation (e.g., one or more of        a deletion, an insertion, a substitution, e.g., a conservative        substitution), e.g., a deletion, at position 26 in the heavy        chain FW1 relative to A11,    -   j) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a substitution, e.g., a T33V mutation in the heavy chain        CDR1 region relative to A11, and has improved (e.g., relative to        A11) binding to and/or neutralization of dengue virus, e.g., to        one or more (e.g., all) of DV-1, DV-2, DV-3, and DV-4,    -   k) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a deletion, at position 26 in the heavy chain FW1 relative        to A11, and has improved (e.g., relative to A11) binding to        and/or neutralization of dengue virus, e.g., to one or more        (e.g., all) of DV-1, DV-2, DV-3, and DV-4, e.g., to DV-4,    -   l) Has both a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a substitution, e.g., a T33V mutation in the heavy chain        CDR1 region relative to A11 and a mutation (e.g., one or more of        a deletion, an insertion, a substitution, e.g., a conservative        substitution), e.g., a deletion, at position 26 in the heavy        chain FW1 relative to A11, and has improved (e.g., relative to        A11) binding to and/or neutralization of dengue virus, e.g., to        one or more (e.g., all) of DV-1, DV-2, DV-3, and DV-4, e.g., to        DV-4,    -   m) Displays improved binding to EDIII of one or more (one or        more DV-1 strains, one or more DV-2 strains, one or more DV-3        strains, one or more DV-4 strains, e.g., one or more of: DENV-4        BC2, DENV-4-Sing10, DENV-4 NewCal09, DENV-4 Phil56, DENV-4        Thailand/1997, DENV-3 Sing09, DENV-3 Nic10, DENV-4 Brazil/2011,        DENV-4 Venezuela/2008, DENV-4 Colombia/1997, DENV-3 H87, DENV-3        Puerto Rico/1977, DENV-3 Cambodia/2008, DENV-2 Peru95, DENV-2        Sing08, DENV-2 NGC, DENV-2 Venezuela/2007, DENV-2 Vietnam/2007,        DENV-1 Hawaii/1944, DENV-2 New Guinea/1944 (NGC), DENV-3        Philippines/1956 (H87), DENV-4 Mexico/1997 (BC287/97), and        DENV-4 H241, e.g., with at least 2, 3, 4, 5, 6, 8, 10, 12, 25,        50, 75, 100, or 1,000-fold greater affinity,    -   n) Disrupts the native structure of the E protein on the surface        of the virion, e.g., which may cause inactivation of the virus,    -   o) Binds specifically to an epitope on EDIII, e.g., the same or        similar epitope as the epitope recognized by a A11 or B11        monoclonal antibody,    -   p) Shows the same or similar binding affinity or specificity, or        both, as an antibody of Table 1, e.g., D88, A48, F38, F108, or        C88,    -   q) Shows the same or similar binding affinity or specificity, or        both, as an antibody molecule (e.g., a heavy chain variable        region and light chain variable region) described in Table 1,        e.g., D88, A48, F38, F108, or C88,    -   r) Shows the same or similar binding affinity or specificity, or        both, as an antibody molecule (e.g., a heavy chain variable        region and light chain variable region) comprising an amino acid        sequence shown in Table 2,    -   s) Inhibits, e.g., competitively inhibits, the binding of a        second antibody molecule to EDIII wherein the second antibody        molecule is an antibody molecule described herein, e.g., an        antibody molecule chosen from Table 1, e.g., D88, A48, F38,        F108, or C88,    -   t) Binds the same or an overlapping epitope with a second        antibody molecule to EDIII, wherein the second antibody molecule        is an antibody molecule described herein, e.g., an antibody        molecule chosen from Table 1, e.g., D88, A48, F38, F108, or C88,    -   u) Competes for binding and binds the same epitope, with a        second antibody molecule to EDIII, wherein the second antibody        molecule is an antibody molecule described herein, e.g., an        antibody molecule chosen from Table 1, e.g., D88, A48, F38,        F108, or C88,    -   v) Has one or more biological properties of an antibody molecule        described herein, e.g., an antibody molecule chosen from Table        1, e.g., D88, A48, F38, F108, or C88,    -   w) Has one or more pharmacokinetic properties of an antibody        molecule described herein, e.g., an antibody molecule chosen        from Table 1, e.g., D88, A48, F38, F108, or C88, or    -   x) Inhibits one of more activities of dengue virus, e.g.,        neutralizes the virus (for instance, measured in a focus        reduction neutralization test or a related test for evaluating        neutralization of viral activity).

In some embodiments, the antibody molecule has a mutation (e.g., one ormore of a deletion, an insertion, a substitution, e.g., a conservativesubstitution), e.g., a T33V mutation in the heavy chain CDR1 regionrelative to A11, in combination with one or more functional propertiesof List 1 above, e.g., one or more (e.g., two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, or all) of properties (a), (b), (c), (d), (e), (m), (n), (o),(p), (q), (r), (s), (t), (u), (v), (w), or (x). In certain embodiments,the antibody molecule has a mutation (e.g., one or more of a deletion,an insertion, a substitution, e.g., a conservative substitution), e.g.,a deletion, at position 26 in the heavy chain FW1 relative to A11, incombination with one or more functional properties of List 1 above,e.g., one or more (e.g., two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all)of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r),(s), (t), (u), (v), (w), or (x). In some embodiments, the antibodymolecule has both a mutation (e.g., one or more of a deletion, aninsertion, a substitution, e.g., a conservative substitution), e.g., aT33V mutation in the heavy chain CDR1 region relative to A11 and amutation (e.g., one or more of a deletion, an insertion, a substitution,e.g., a conservative substitution), e.g., a deletion, at position 26 inthe heavy chain FW1 relative to A11, in combination with one or morefunctional properties of List 1 above, e.g., one or more (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In some embodiments, the antibody molecule has a T33V mutation in theheavy chain CDR1 region relative to A11, in combination with one or morefunctional properties of List 1 above, e.g., one or more (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x). In certain embodiments, the antibody molecule has a deletion atposition 26 in the heavy chain FW1 relative to A11, in combination withone or more functional properties of List 1 above, e.g., one or more(e.g., two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, or all) of properties (a),(b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v),(w), or (x). In some embodiments, the antibody molecule has both a T33Vmutation in the heavy chain CDR1 region relative to A11 and a deletionat position 26 in the heavy chain FW1 relative to A11, in combinationwith one or more functional properties of List 1 above, e.g., one ormore (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all) ofproperties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s),(t), (u), (v), (w), or (x).

In certain embodiments, the antibody molecule has both a T33V mutationin the heavy chain CDR1 region relative to A11 and a deletion, atposition 26 in the heavy chain FW1 relative to A11, combination with oneor more functional properties of List 1 above, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w),(x). For example, the antibody molecule may bind EDIII (e.g., of DV-1,DV-2, DV-3, or DV-4) with high affinity, e.g., with a dissociationconstant (K_(D)) of less than about 100 nM, typically about 10 nM, andmore typically, about 10-0.01 nM, about 5-0.01 nM, about 3-0.05 nM, orabout 1-0.1 nM, or stronger, e.g., less than about 80, 70, 60, 50, 40,30, 20, 10, 8, 6, 4, 3, 2, 1, 0.5, 0.2, 0.1, 0.05, or 0.01 nM. As afurther example, the antibody molecule may bind to DV-4 EDIII with highaffinity, e.g., with a dissociation constant (K_(D)) of less than about100 nM, e.g., about 10 nM, e.g., about 10-1 nM or stronger, e.g., lessthan about 10, 8, 6, 5, 4, or 3 nM. Furthermore, the antibody moleculemay neutralize DV-4 with an improved IC50 compared to antibody A11and/or antibody 4E11, e.g., at least 2, 3, 4, 5, 6, 8, 10, 12, 100,1,000-fold improved IC50, e.g., in a focus reduction neutralization testor a related test for evaluating neutralization of viral activity.

In certain embodiments, affinity is measured by competition ELISA orSPR. In some embodiments, affinity is measured by one or more ofBIAcore, ELISA, or flow cytometry.

In certain embodiments, the anti-dengue antibody molecule is a humanizedantibody molecule and has one or more properties from List 1 above,e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, or all) of properties (a), (b), (c), (d), (e), (m), (n), (o),(p), (q), (r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the antibody molecule binds to EDIII with highaffinity, e.g., with a K_(D) that is at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80% or 90% lower than the K_(D) of a murine anti-dengueantibody molecule, e.g., 4E11, A11 or B11.

In some embodiments, the expression level of the antibody molecule ishigher, e.g., at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-foldhigher, than the expression level of a murine antibody molecule, e.g., amurine anti-dengue antibody molecule described herein. In someembodiments, the antibody molecule is expressed in mammalian cells,e.g., human or rodent cells.

In some embodiments, the antibody molecule reduces one or more denguevirus activities with an IC50 (concentration at 50% inhibition) that islower, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or90% lower, than the IC50 of a murine anti-dengue antibody molecule,e.g., a murine anti-dengue antibody molecule described herein. In someembodiments, the dengue virus activity is neutralized, e.g., in thefocus reduction neutralization test described in Tharakaramana et al.,Proc Natl Acad Sci USA. 2013 Apr. 23; 110(17):E1555-64. doi:10.1073/pnas.1303645110. Epub 2013 Apr. 8, which is hereby expresslyincorporated by reference in its entirety, including all supplementalmaterials. Other related tests that can be used to evaluateneutralization of viral activity include, e.g., enzyme-linkedimmunosorbent assay (ELISA)-based microneutralization (MN) assays (e.g.,as described in Vorndam et al., Am J Trop Med Hyg 2002; 66: 208-212) andfluorescent antibody cell sorter-based, DC-SIGN expresser dendritic cell(DC) assay (e.g., as described in Martin et al., J Virol Methods 2006;134: 74-85).

In certain embodiments, the antibody molecule reduces transmission ofdengue virus (e.g., reduces transmission between a subject (e.g., ahuman) and a mosquito), e.g., by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90%, e.g., as measured by a mosquito model describedherein.

In other embodiments, the antibody molecule has an improved ability toreduce transmission of dengue virus (e.g., has an improved ability toreduce transmission of dengue virus between a subject (e.g., a human)and a mosquito), e.g., by at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10-fold higher than a murine anti-dengue antibody molecule, e.g., amurine anti-dengue antibody molecule described herein, e.g., 4E11, A11or B11, e.g., as measured by a mosquito model described herein.

In other embodiments, the antibody molecule reduces the mosquito viralload (e.g., the amount of virus, and/or infectivity, carried by amosquito), e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90%, e.g., as measured by a mosquito model described herein.

In some embodiments, the antibody molecule has improved stability, e.g.,at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold more stable invivo or in vitro, than a murine anti-dengue antibody molecule, e.g., amurine anti-dengue antibody molecule described herein, e.g., 4E11, A11or B11.

In some embodiments, the anti-dengue antibody molecule comprises atleast one antigen-binding region, e.g., a variable region or anantigen-binding fragment thereof, from an antibody described herein,e.g., an antibody chosen from Table 1, e.g., D88, A48, F38, F108, orC88, or a sequence substantially identical to any of the aforesaidsequences (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, and/or having one, two, three or more substitutions,insertions or deletions, e.g., conserved substitutions). In someembodiments, an antibody molecule has a structural feature discussed inthis paragraph and one or more advantageous properties such as animproved (e.g., relative to A11) affinity for or neutralization activitytowards dengue virus, e.g., DV-4. In some embodiments, the advantageousproperty is a property of List 1, e.g., one or more (e.g., two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, or all) of properties (a), (b), (c), (d),(e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises atleast one, two, three, or four variable regions from an antibodydescribed herein, e.g., an antibody of Table 1, e.g., D88, A48, F38,F108, or C88, or a sequence substantially identical to any of theaforesaid sequences (e.g., a sequence at least about 85%, 90%, 95%, 99%or more identical thereto, and/or having one, two, three or moresubstitutions, insertions or deletions, e.g., conserved substitutions).In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In some embodiments, the anti-dengue antibody molecule comprises atleast one or two heavy chain variable regions from an antibody describedherein, e.g., an antibody of Table 1, e.g., D88, A48, F38, F108, or C88,or a sequence substantially identical to any of the aforesaid sequences(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, and/or having one, two, three or more substitutions, insertionsor deletions, e.g., conserved substitutions). In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises atleast one or two light chain variable regions from an antibody describedherein, e.g., an antibody of Table 1, e.g., D88, A48, F38, F108, or C88or a sequence substantially identical to any of the aforesaid sequences(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, and/or having one, two, three or more substitutions, insertionsor deletions, e.g., conserved substitutions). In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises avaline at position 33 (e.g., a T33V mutation) in the VH region, e.g.,relative to 4E5A and/or 4E11 or an antibody of Table 1. In someembodiments, the anti-dengue antibody comprises a del26 (deletion atposition 26) in the VH region, e.g., relative to 4E5A or an antibody ofTable 1. In some embodiments, the anti-dengue antibody comprises both avaline at position 33 (e.g., a T33V mutation) and a del26 mutation inthe VH region, e.g., relative to 4E5A or an antibody of Table 1. In someembodiments, an antibody molecule has a structural feature discussed inthis paragraph and one or more advantageous properties such as animproved (e.g., relative to A11) affinity for or neutralization activitytowards dengue virus, e.g., DV-4. In some embodiments, the advantageousproperty is a property of List 1, e.g., one or more (e.g., two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, or all) of properties (a), (b), (c), (d),(e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule comprises atleast one, two, or three complementarity determining regions (CDRs) froma heavy chain variable region of an antibody described herein, e.g., anantibody of Table 1, e.g., D88, A48, F38, F108, or C88, or a sequencesubstantially identical to any of the aforesaid sequences (e.g., asequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or having one, two, three or more substitutions, insertions ordeletions, e.g., conserved substitutions). In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from aheavy chain variable region comprising an amino acid sequence shown inTable 3. In some embodiments, one or more of the CDRs (or collectivelyall of the CDRs) have one, two, three, four, five, six or more changes,e.g., amino acid substitutions, insertions, or deletions, relative tothe CDRs shown in Table 3. In some embodiments, an antibody molecule hasa structural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three CDRs (or collectively all of the CDRs) from a lightchain variable region comprising an amino acid sequence shown in Table3. In some embodiments, one or more of the CDRs (or collectively all ofthe CDRs) have one, two, three, four, five, six or more changes, e.g.,amino acid substitutions, insertions, or deletions, relative to the CDRsshown in Table 3. In some embodiments, an antibody molecule has astructural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, three, four, five or six CDRs (or collectively all of theCDRs) from a heavy and light chain variable region comprising an aminoacid sequence shown in Table 3. In some embodiments, one or more of theCDRs (or collectively all of the CDRs) have one, two, three, four, five,six or more changes, e.g., amino acid substitutions, insertions, ordeletions, relative to the CDRs shown in Table 3. In some embodiments,an antibody molecule has a structural feature discussed in thisparagraph and one or more advantageous properties such as an improved(e.g., relative to A11) affinity for or neutralization activity towardsdengue virus, e.g., DV-4. In some embodiments, the advantageous propertyis a property of List 1, e.g., one or more (e.g., two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule includes allsix CDRs from an antibody described herein, e.g., an antibody of Table1, e.g., D88, A48, F38, F108, or C88, or closely related CDRs, e.g.,CDRs which are identical or which have at least one amino acidalteration, but not more than two, three or four alterations (e.g.,substitutions e.g., conservative substitutions, deletions, orinsertions). In certain embodiments, the anti-dengue antibody moleculemay include any CDR described herein. In some embodiments, an antibodymolecule has a structural feature discussed in this paragraph and one ormore advantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three Chothia hypervariable loops from a heavy chainvariable region of an antibody described herein, e.g., an antibody ofTable 1, e.g., D88, A48, F38, F108, or C88, or at least the amino acidsfrom those hypervariable loops that contact EDIII. For instance, in someembodiments, an antibody molecule provided herein has a VHCDR1 of SEQ IDNO: 9, a VHCDR2 of SEQ ID NO: 10, and a VHCDR3 of SEQ ID NO: 5. Anantibody molecule provided herein may also have a VHCDR1 of SEQ ID NO:15, a VHCDR2 of SEQ ID NO: 10, and a VHCDR3 of SEQ ID NO: 5. An antibodymolecule provided herein may also have a VHCDR1 of SEQ ID NO: 22, 24,26, 28, or 30; a VHCDR2 of SEQ ID NO: 10; and a VHCDR3 of SEQ ID NO: 5.In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In some embodiments, and optionally in combination with heavy chain CDRsdescribed herein, the anti-dengue antibody molecule includes at leastone, two, or three Chothia hypervariable loops from a light chainvariable region of an antibody described herein, e.g., an antibody ofTable 1, e.g., D88, A48, F38, F108, or C88, or at least the amino acidsfrom those hypervariable loops that contact EDIII. For instance, incertain embodiments, an antibody molecule provided herein has a VHCDR1of SEQ ID NO: 6, a VHCDR2 of SEQ ID NO: 7, and a VHCDR3 of SEQ ID NO: 8.In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three Kabat hypervariable loops from a heavy chain variableregion of an antibody described herein, e.g., an antibody of Table 1,e.g., D88, A48, F38, F108, or C88, or at least the amino acids fromthose hypervariable loops that contact EDIII. In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three Kabat hypervariable loops from a light chain variableregion of an antibody described herein, e.g., an antibody of Table 1,e.g., D88, A48, F38, F108, or C88, or at least the amino acids fromthose hypervariable loops that contact EDIII. In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule includes atleast one, two, three, four, five, or six hypervariable loops from theheavy and light chain variable regions of an antibody described herein,e.g., an antibody of Table 1, e.g., D88, A48, F38, F108, or C88, or atleast the amino acids from those hypervariable loops that contact EDIII.In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In certain embodiments, the anti-dengue antibody molecule includes allsix hypervariable loops from the heavy and light chain variable regionsof an antibody described herein, e.g., an antibody of Table 1, e.g.,D88, A48, F38, F108, or C88, or at least the amino acids from thosehypervariable loops that contact EDIII, or closely related hypervariableloops, e.g., hypervariable loops which are identical or which have atleast one amino acid alteration, but not more than two, three or fouralterations (e.g., substitutions, e.g., conservative substitutions,deletions, or insertions). In some embodiments, an antibody molecule hasa structural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three hypervariable loops that have the same canonicalstructures as the corresponding hypervariable loop of an antibodydescribed herein, e.g., an antibody of Table 1, e.g., D88, A48, F38,F108, or C88, e.g., the same canonical structures as at least loop 1and/or loop 2 of the heavy and/or light chain variable domains of anantibody described herein. See, e.g., Chothia et al., (1992) J. Mol.Biol. 227.-799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798for descriptions of hypervariable loop canonical structures. Thesestructures can be determined by inspection of the tables described inthese publications. In some embodiments, an antibody molecule has astructural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises atleast one, two, or three (e.g., all) CDRs from a heavy chain variableregion having an amino acid sequence as set forth in Table 3, or asequence substantially identical thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or having one,two, three or more substitutions, insertions or deletions, e.g.,conserved substitutions). In some embodiments, the anti-dengue antibodymolecule comprises at least one, two, or three (e.g., all) CDRs from alight chain variable region having an amino acid sequence as set forthin Table 3, or a sequence substantially identical thereto (e.g., asequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or having one, two, three or more substitutions, insertions ordeletions, e.g., conserved substitutions). In certain embodiments, theanti-dengue antibody molecule comprises at least one, two, three, four,five or six (e.g., all) CDRs from heavy and light chain variable regionshaving an amino acid sequence as set forth in Table 3, or a sequencesubstantially identical thereto (e.g., a sequence at least about 85%,90%, 95%, 99% or more identical thereto, and/or having one, two, threeor more substitutions, insertions or deletions, e.g., conservedsubstitutions). In some embodiments, an antibody molecule has astructural feature discussed in this paragraph and one or moreadvantageous properties such as an improved affinity for orneutralization activity towards dengue virus, e.g., DV-4. In someembodiments, the advantageous property is a property of List 1, e.g.,one or more (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all) ofproperties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s),(t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule comprises atleast one, two, or three (e.g., all) CDRs from a heavy chain variableregion having an amino acid sequence of an antibody described herein,e.g., an antibody of Table 1, e.g., D88, A48, F38, F108, or C88, or asequence substantially identical thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or having one,two, three or more substitutions, insertions or deletions, e.g.,conserved substitutions). In certain embodiments, the anti-dengueantibody molecule comprises at least one, two, or three (e.g., all) CDRsfrom a light chain variable region having an amino acid sequence of anantibody described herein, e.g., an antibody of Table 1, e.g., D88, A48,F38, F108, or C88, or a sequence substantially identical thereto (e.g.,a sequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or having one, two, three or more substitutions, insertions ordeletions, e.g., conserved substitutions). In some embodiments, theanti-dengue antibody molecule comprises six CDRs described herein, e.g.,in a VL and VH sequence of Table 2. In some embodiments, an antibodymolecule has a structural feature discussed in this paragraph and one ormore advantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In aspects embodiments, the antibody molecule has a VHCDR1, VHCDR2,VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from Table 3. The six CDRsmay all be Kabat-defined, all Chothia-defined, or some Kabat- and someChothia-defined. For instance, VHCDR1 may be selected from SEQ ID NO: 3,9, 14, 15, 22, 24, 26, 28, or 30; VHCDR2 may be selected from SE$Q IDNO: 4, 10, or 35; VHCDR3 may be SEQ ID NO: 5; VLCDR1 may be SEQ ID NO:6; VLCDR2 may be SEQ ID NO: 7; and VLCDR3 may be SEQ ID NO: 8.

In certain embodiments, the light or the heavy chain variable frameworkof the anti-dengue antibody can be chosen from: (a) a light or heavychain variable framework including at least 80%, 85%, 87% 90%, 92%, 93%,95%, 97%, 98%, or preferably 100% of the amino acid residues from ahuman light or heavy chain variable framework, e.g., a light or heavychain variable framework residue from a human mature antibody, a humangermline sequence, or a human consensus sequence; (b) a light or heavychain variable framework including from 20% to 80%, 40% to 60%, 60% to90%, or 70% to 95% of the amino acid residues from a human light orheavy chain variable framework, e.g., a light or heavy chain variableframework residue from a human mature antibody, a human germlinesequence, or a human consensus sequence; (c) a non-human framework(e.g., a rodent framework); or (d) a non-human framework that has beenmodified, e.g., to remove antigenic or cytotoxic determinants, e.g.,de-immunized, or partially humanized. In some embodiments, the light orheavy chain variable framework region includes a light or heavy chainvariable framework sequence at least 70, 75, 80, 85, 87, 88, 90, 92, 94,95, 96, 97, 98, 99% identical or identical to the frameworks of a VH orVL segment of a human germline gene. In some embodiments, an antibodymolecule has a structural feature discussed in this paragraph and one ormore advantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the VH region (e.g., the framework regionstherein) of the anti-dengue antibody comprises one or more positionsfrom a human VH region, e.g., human heavy chain germline-encoded aminoacid sequences, e.g., positions found in one or more (e.g., all) of FW1,FW2, FW3, and FW4. In certain embodiments, optionally in combinationwith the VH residues discussed in the previous sentence, the VL region(e.g., the framework regions therein) of the anti-dengue antibodycomprises one or more positions from a human VL region, e.g., humanheavy chain germline-encoded amino acid sequences, e.g., positions foundin one or more (e.g., two, three, four, five, or all) of FW1, FW2, FW3,and FW4.

For example, in some embodiments, the antibody molecule comprises one ormore (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or all)residues according to heavy chain or light chain FW1, FW2, FW3, or FW4regions from a human germline sequence of Table 5. More specifically, insome embodiments the antibody molecule has one or more (e.g., at least2, 3, 4, 5, 10, or 15, or all) VH FW1 residues of a VH germline sequenceof Table 6; in some embodiments the antibody molecule has one or more(e.g., at least 2, 3, 4, 5, 10, or 15, or all) VH FW2 residues of a VHgermline sequence of Table 6; in some embodiments the antibody moleculehas one or more (e.g., at least 2, 3, 4, 5, 10, or 15, or all) VH FW3residues of a VH germline sequence of Table 6, and in some embodimentsthe antibody molecule has one or more (e.g., at least 2, 3, 4, 5, 10, or15, or all) VH FW4 residues of a VH germline sequence of Table 6.Furthermore, and optionally in combination with the heavy chain residuesdiscussed in the previous sentence, in some embodiments the antibodymolecule has one or more (e.g., at least 2, 3, 4, 5, 10, or 15, or all)VL FW1 residues of a VL germline sequence of Table 6; in someembodiments the antibody molecule has one or more (e.g., at least 2, 3,4, 5, 10, or 15, or all) VL FW2 residues of a VL germline sequence ofTable 6; in some embodiments the antibody molecule has one or more(e.g., at least 2, 3, 4, 5, 10, or 15, or all) VL FW3 residues of a VLgermline sequence of Table 6, and in some embodiments the antibodymolecule has one or more (e.g., at least 2, 3, 4, 5, 10, or 15, or all)VL FW4 residues of a VL germline sequence of Table 6. In certainembodiments, the antibody molecule has a heavy chain frameworkVH1-18*01, JH4*01 and/or light chain framework Vk4-1*01, Jk2*02.

In certain embodiments, the anti-dengue antibody molecule comprises aheavy chain variable domain having at least one, two, three, four, five,six, seven, ten, fifteen, twenty or more changes, e.g., amino acidsubstitutions, insertions, or deletions, from an amino acid sequence ofTable 1, e.g., B11, D88, A48, F38, F108, or C88, e.g., the amino acidsequence of the FR region in the entire variable region. In someembodiments, the anti-dengue antibody molecule comprises a heavy chainvariable domain having one or more (e.g., at least 5, 10, 15, or 20, orall) of: Q at position 3, V at position 5, a deletion of E at position6, V at position 12, K at position 13, K at position 20, V at position21, K at position 24, a deletion of S at position 26, V at position 33,R at position 39, A at position 41, G at position 43, M at position 49,L at position 65, R at position 68, V at position 69, M at position 71,T at position 77, M at position 82, E at position 83, R at position 85,R at position 88, D at position 90, A or V or S at position 98, and S atposition 117 of the amino acid sequence of an antibody of Table 1, e.g.,A11. Examples of antibodies having one or more (e.g., all) of thesemutations include A48, B48, C88, F38, F108, and D48. In someembodiments, the humanized heavy chain contains one or more of: Q atposition 3, V at position 5, a deletion of E at position 6, V atposition 12, K at position 13, K at position 20, V at position 21, K atposition 24, R at position 39, A at position 41, G at position 43, M atposition 49, L at position 65, R at position 68, V at position 69, M atposition 71, T at position 77, M at position 82, E at position 83, R atposition 85, R at position 88, D at position 90, V or A or S at position98, and S at position 117 of the amino acid sequence of an antibody ofTable 1, e.g., A11. An example of an antibody having one or more (e.g.,all) of these mutations is A68. In some embodiments, an antibodymolecule has a structural feature discussed in this paragraph and one ormore advantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In certain embodiments (and optionally in combination with the heavychain substitutions described herein, e.g., in the previous paragraph),the anti-dengue antibody molecule comprises a light chain variabledomain having at least one, two, three, four, five, six, seven, ten,fifteen, twenty or more amino acid changes, e.g., amino acidsubstitutions, insertions, or deletions, from an amino acid sequence ofTable 1, e.g., B11, D88, A48, F38, F108, or C88, e.g., the amino acidsequence of the FR region in the entire variable region. In certainembodiments, the anti-dengue antibody comprises a light chain variabledomain having one or more (e.g., at least 5, 10, 15, or all) of: D atposition 1, I at position 2, S at position 7, E at position 17, P atposition 44, V at position 62, D at position 64, G at position 72, S atposition 80, S at position 81, L at position 82, Q at position 83, E atposition 85, V at position 89, Y at position 91, and Q at position 104of the amino acid sequence of an antibody of Table 1, e.g., B11, D88,A48, F38, F108, or C88. In some embodiments, an antibody molecule has astructural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the heavy or light chain variable domain, or both,of the of the anti-dengue antibody molecule includes an amino acidsequence, which is substantially identical to an amino acid disclosedherein, e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higheridentical to a variable region of an antibody described herein, e.g., anantibody of Table 1, e.g., D88, A48, F38, F108, or C88, or which differsby at least 1, 2, 3, 4, or 5 residues, but less than 40, 30, 20, or 10residues, from a variable region of an antibody described herein. Insome embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In certain embodiments, the heavy or light chain variable region, orboth, of the anti-dengue antibody molecule includes an amino acidsequence encoded by a nucleic acid sequence described herein, or anucleic acid that hybridizes to a nucleic acid sequence that encodes anantibody of Table 1, e.g., D88, A48, F38, F108, or C88, or itscomplement, e.g., under low stringency, medium stringency, or highstringency, or other hybridization condition described herein. Thisapplication also discloses the heavy or light chain variable region, orboth, of the anti-dengue antibody molecule includes an amino acidsequence encoded by a nucleic acid sequence of Table 4, or itscomplement, e.g., under low stringency, medium stringency, or highstringency, or other hybridization condition described herein. In someembodiments, the nucleic acid is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or higher identical to a sequence of Table 4 or a portionthereof. In some embodiments, an antibody molecule has a structuralfeature discussed in this paragraph and one or more advantageousproperties such as an improved (e.g., relative to A11) affinity for orneutralization activity towards dengue virus, e.g., DV-4. In someembodiments, the advantageous property is a property of List 1, e.g.,one or more (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all) ofproperties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s),(t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises atleast one, two, three, or four antigen-binding regions, e.g., variableregions, having an amino acid sequence as set forth in Table 2, or asequence substantially identical thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, or which differs byno more than 1, 2, 5, 10, or 15 amino acid residues from the sequencesshown in Table 2). In certain embodiments, the anti-dengue antibodymolecule includes a VH and/or VL domain encoded by a nucleic acid havinga nucleotide sequence that encodes an antibody of Table 1, e.g., D88,A48, F38, F108, or C88, or a sequence substantially identical to any oneof the nucleotide sequences (e.g., a sequence at least about 85%, 90%,95%, 99% or more identical thereto, or which differs by no more than 3,6, 15, 30, or 45 nucleotides from any one of the nucleotide sequences).In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In certain aspects, the present disclosure provides an antibodymolecule, optionally capable of binding dengue virus, comprising:

(a) a heavy chain immunoglobulin variable region segment comprising:

a CDR1 comprising the sequence DVYMS (SEQ ID NO: 3) (or a sequence thatdiffers by no more than, 1, 2, or 3 amino acids therefrom, optionallyprovided that V is unchanged),

a CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom, optionally provided that L is unchanged), and

a CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom);

(b) a light chain variable region segment comprising:

a CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom),

a CDR2 comprising the sequence RASELQW (SEQ ID NO: 7) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom), and

a CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom).

In some embodiments, V of HCDR1 is unchanged; in some embodiments, L ofHCDR2 is unchanged, and in some embodiments, both V of HCDR1 and L ofHCDR2 are unchanged.

In certain embodiments, the antibody molecule comprises a VH FW1 havingthe sequence QVQLVQSGAEVKKPGASVKVSCKAGFNIK (SEQ ID NO: 11), or an aminoacid sequence having no more than 1, 2, 3, 4, or 5 mutations relative toSEQ ID NO: 11.

In certain embodiments, the antibody molecule comprises a VH FW2 havingthe sequence WVRQAPGQGLEWMG (SEQ ID NO: 84), or an amino acid sequencehaving no more than 1, 2, 3, 4, or 5 mutations relative to SEQ ID NO:84. In certain embodiments, the antibody molecule comprises a VH FW2having the sequence WVRQAPEQGLEWMG (SEQ ID NO: 85), or an amino acidsequence having no more than 1, 2, 3, 4, or 5 mutations relative to SEQID NO: 85.

In certain aspects, the present disclosure provides an antibody moleculecapable of binding dengue virus, comprising:

(a) a heavy chain immunoglobulin variable region segment comprising:

a FW1 comprising a deletion of position 26 relative to SEQ ID NO: 33;

a CDR1 comprising the sequence DTYMS (SEQ ID NO: 14) (or a sequence thatdiffers by no more than, 1, 2, or 3 amino acids therefrom, optionallyprovided that T is unchanged), or a CDR1 comprising the sequence DVYMS(SEQ ID NO: 3) (or a sequence that differs by no more than, 1, 2, or 3amino acids therefrom, optionally provided that V is unchanged),

a CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom, optionally provided that L is unchanged), and

a CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom); and

(b) a light chain variable region segment comprising:

a CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom),

a CDR2 comprising the sequence RASELQW (SEQ ID NO: 7) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom), and

a CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom).

In certain embodiments, the antibody molecule comprises a VH FW2 havingthe sequence WVRQAPGQGLEWMG (SEQ ID NO: 84), or an amino acid sequencehaving no more than 1, 2, 3, 4, or 5 mutations relative to SEQ ID NO:84. In certain embodiments, the antibody molecule comprises a VH FW2having the sequence WVRQAPEQGLEWMG (SEQ ID NO: 85), or an amino acidsequence having no more than 1, 2, 3, 4, or 5 mutations relative to SEQID NO: 85.

In certain aspects, the present disclosure provides an antibodymolecule, optionally capable of binding dengue virus, comprising:

(a) a heavy chain immunoglobulin variable region segment comprising:

a CDR1 comprising the sequence DVYMS (SEQ ID NO: 3) (or a sequence thatdiffers by no more than, 1, 2, or 3 amino acids therefrom, optionallyprovided that V is unchanged),

a CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom, optionally provided that L is unchanged), and

a CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom),optionally provided that A is replaced with I, K, D or E;

(b) a light chain variable region segment comprising:

a CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom), optionally provided at Y is replaced with F,

a CDR2 comprising the sequence RASELQW (SEQ ID NO: 7) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom), and

a CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom).

Accordingly, in some embodiments, the heavy chain CDR3 is GWEGFIY (SEQID NO: 90), GWEGFKY (SEQ ID NO: 91), GWEGFDY (SEQ ID NO: 92), or GWEGFEY(SEQ ID NO: 93). In some embodiments, the light chain CDR1 isRASENVDKFGNSFMH (SEQ ID NO: 94). Put another way, in some embodiments,position 105 of the heavy chain, which is alanine in antibody A11, maybe changed to another residue, e.g., an I, K, D or E. In certainembodiments, position 32 of the light chain, which is tyrosine inantibody A11, is changed to another residue, e.g., F. In someembodiments, a mutation described in this paragraph improves theantibody molecule's affinity for EDIII and/or its neutralizationactivity towards one or more (or all) strain or serotype of denguevirus.

In certain embodiments, the antibody molecule comprises a VH FW1 havingthe sequence QVQLVQSGAEVKKPGASVKVSCKAGFNIK (SEQ ID NO: 11), or an aminoacid sequence having no more than 1, 2, 3, 4, or 5 mutations relative toSEQ ID NO: 11.

In certain embodiments, the antibody molecule comprises a VH FW2 havingthe sequence WVRQAPGQGLEWMG (SEQ ID NO: 84), or an amino acid sequencehaving no more than 1, 2, 3, 4, or 5 mutations relative to SEQ ID NO:84. In certain embodiments, the antibody molecule comprises a VH FW2having the sequence WVRQAPEQGLEWMG (SEQ ID NO: 85), or an amino acidsequence having no more than 1, 2, 3, 4, or 5 mutations relative to SEQID NO: 85.

In some embodiments of the aspects herein, the antibody molecule iscapable of binding to dengue virus EDIII (E protein domain III). Incertain embodiments, the antibody molecule comprises one or more CDRshaving the sequence of any of SEQ ID NOS: 3-8, 14, and 35 (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom). Forexample, the antibody molecule may comprise at least two, three, four,five, or six CDRs having the sequence of any of SEQ ID NOS: 3-8, 14, and35.

In some embodiments, the antibody molecule comprises a VH CDR1 of SEQ IDNO: 3 or 14, a VH CDR2 of SEQ ID NO: 4 or 35, a VH CDR3 of SEQ ID NO: 5,a VL CDR1 of SEQ ID NO: 6, a VL CDR2 of SEQ ID NO: 7, and a VL CDR3 ofSEQ ID NO: 8. For instance, the antibody molecule may comprise a VH CDR1of SEQ ID NO: 3, a VH CDR2 of SEQ ID NO: 4, a VH CDR3 of SEQ ID NO: 5, aVL CDR1 of SEQ ID NO: 6, a VL CDR2 of SEQ ID NO: 7, and a VL CDR3 of SEQID NO: 8.

In some embodiments, the antibody molecule comprises a VH amino acidsequence at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQID NO: 1.

In some embodiments, the antibody molecule comprises a VH amino acidsequence at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQID NO: 80.

In some embodiments, the antibody molecule comprises a VH amino acidsequence at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to anyof SEQ ID NOs. 16-21, 24, 25, 27, 29, 31, 32, 33, 36, 80, or 81. In someembodiments, the antibody molecule comprises a VL amino acid sequence atleast 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2 or34.

In certain embodiments, the antibody molecule is a Fab, F(ab′)2, Fv, ora single chain Fv fragment (scFv). In some embodiments, the antibodymolecule comprises a heavy chain constant region selected from IgG1,IgG2, IgG3, and IgG4. In some embodiments, the antibody moleculecomprises a light chain constant region chosen from the light chainconstant regions of kappa or lambda.

The antibody molecule may be an isolated antibody molecule and/or ahumanized antibody molecule. In some embodiments, the antibody moleculecontains one or more framework regions derived from a human frameworkgermline sequence.

In some embodiments, the antibody molecule is capable of binding todengue virus EDIII with a dissociation constant (K_(D)) of less thanabout 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4, 3, 2, 1, 0.5, 0.2, or 0.1nM. The antibody molecule may be capable of binding to dengue virusserotype DV-4 EDIII with a dissociation constant (K_(D)) of less thanabout 10, 8, 6, 5, 4, or 3 nM. The antibody molecule may be capable ofbinding to DV-3 or DV-4 EDIII domain with at least a 2, 3, 4, 5, 6, 8,10, 12, 100, 1,000-fold greater affinity than antibody A11 or antibody4E11. The antibody molecule may be capable of binding to a dengue virusstrain chosen from one or more of DENV-4 BC2, DENV-4-Sing10, DENV-4NewCal09, DENV-4 Phil56, DENV-3 Sing09, DENV-3 Nic10, DENV-3 H87, DENV-2Peru95, DENV-2 Sing08, DENV-2 NGC, DENV-1 Hawaii/1944, DENV-2 NewGuinea/1944 (NGC), DENV-3 Philippines/1956 (H87), DENV-4 Mexico/1997(BC287/97), and DENV-4 H241, with at least 2, 3, 4, 5, 6, 8, 10, 12, 25,50, 75, 100, or 1,000-fold greater affinity than antibody A11 orantibody 4E11. The antibody molecule may be capable of neutralizingdengue virus in a focus reduction neutralization test or a related testfor evaluating neutralization of viral activity. The antibody moleculemay be capable of neutralizing dengue virus with an IC50 that is atleast 2, 3, 4, 5, 6, 8, 10, 12, 50, 75, or 100-fold lower than antibodyA11 or antibody 4E11 in a focus reduction neutralization test or arelated test for evaluating neutralization of viral activity.

In some aspects, the present disclosure provides a formulation, e.g.,pharmaceutical formulation or composition, comprising the antibodymolecule as described herein and a pharmaceutically acceptable carrier,excipient, or stabilizer, e.g., as described herein.

The present disclosure also provides, e.g., a nucleic acid encoding theantibody heavy or light chain variable region of an antibody molecule asdescribed herein. The disclosure also provides, for example, expressionvector comprising such a nucleic acid. The disclosure also provides, forexample, a host cell comprising such a nucleic acid. The presentdisclosure additionally provides, e.g., a method of producing anantibody molecule or fragment thereof as described herein, comprisingculturing the host cell under conditions suitable for gene expression.

In some aspects, this disclosure provides a kit comprising aformulation, e.g., pharmaceutical formulation or composition, comprisingan antibody molecule as described herein. The kit may comprise acontainer, and the container may have the antibody molecule disposedtherein. The kit may also comprise a pharmaceutically acceptablecarrier, excipient, or stabilizer, optionally admixed with the antibodymolecule. The kit may also comprise a delivery device, e.g., onecomprising a syringe or needle. The kit may also comprise instructionsfor use.

In certain aspects, the present disclosure provides a method ofneutralizing dengue virus, comprising: contacting the dengue virus witha formulation as described herein that includes an antibody molecule asdescribed herein. In some embodiments, the dengue virus is of serotypeDV-1, DV-2, DV-3, or DV-4.

In some aspects, the present disclosure provides a method of treating adengue virus infection, comprising administering to a subject in needthereof a formulation as described herein that includes an isolatedantibody molecule as described herein, in an amount effective to treatthe virus. The method may further comprise administering an anti-viralagent to the subject, e.g., an anti-viral agent chosen from one or moreof balapiravir, chloroquine, celgosivir, ivermectin, or Carica folia. Incertain embodiments, the antiviral agent is a second anti-dengueantibody molecule, e.g., an anti-dengue antibody molecule describedherein different from a first anti-dengue antibody molecule. In otherembodiments, the antiviral agent is selected from an alpha-glucosidase Iinhibitor (e.g., celgosivir), an adenosine nucleoside inhibitor (e.g.,NITD008); an RNA-dependent RNA polymerase (RdRp) inhibitor (e.g.,NITD107), an inhibitor of host pyrimidine biosynthesis, e.g., hostdihydroorotate dehydrogenase (DHODH) (e.g., NITD-982 and brequinar), aninhibitor of viral NS4B protein (e.g., NITD-618), and an iminosugar(e.g., UV-4). The method may further comprise administering a vaccine tothe subject, e.g., a dengue virus vaccine. In some embodiments,administration of the antibody molecule is parenteral or intravenous.

The disclosure also provides prophylactic methods. In some embodiments,a method of preventing a dengue virus infection by administering aformulation as described herein that includes an antibody molecule asdisclosed herein to a subject who is not, at the time, infected withdengue virus. For instance, in certain aspects, the present disclosureprovides a method of reducing a patient's risk of contracting denguevirus, comprising administering to a subject in need thereof aformulation as described herein that includes an isolated antibodymolecule as described herein, in an amount effective to reduce the riskof contracting the virus. For example the risk of contracting denguevirus may be reduced by, e.g., at least 25%, 50%, 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or more. In some embodiments, the antibody moleculeis provided to a patient who is not infected with dengue virus, with theresult that if infection occurs, the course of the disease is likely tobe milder than the course of disease in a similar patient who has notreceived the antibody molecule. For instance, the antibody molecule mayreduce the risk of dengue fever developing (e.g., the patient is morelikely to experience an asymptomatic infection). The risk of denguefever developing may be reduced, e.g., by at least 25%, 50%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or more compared to a patient that did notreceive the antibody molecule. In some embodiments, the risk of denguefever progressing into dengue hemorrhagic fever may be reduced, e.g., byat least 25%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or morecompared to a patient that did not receive the antibody molecule.

The disclosure also provides methods of reducing or preventingtransmission of dengue virus (e.g., reducing or preventing transmissionbetween a subject (e.g., a human) and a mosquito. In certainembodiments, the subject is infected with dengue virus. In otherembodiments, the subject is not, at the time, infected with denguevirus, but is at risk of dengue viral infection. For instance, incertain aspects, the present disclosure provides a method of reducing orpreventing transmission of dengue virus (e.g., reducing or preventingtransmission of dengue virus between a subject (e.g., human) and amosquito), comprising administering to a subject a formulation asdescribed herein that includes an isolated antibody molecule asdescribed herein, in an amount effective to reduce the transmission ofdengue virus. For example, the transmission of dengue virus, e.g., froma subject to a mosquito, can be reduced by, e.g., at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to the transmissionfrom a subject that did not receive the antibody molecule, e.g., asmeasured by a mosquito model described herein. As a result, in someembodiments, the transmission of dengue virus from an infected mosquitoto an uninfected subject (e.g., human) can be further reduced, e.g., atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

In certain aspects, this disclosure provides a method of detectingdengue virus in a biological sample, comprising (i) contacting thesample or the subject (and optionally, a reference sample or subject)with an antibody molecule described herein (e.g., in a formulationdescribed herein) under conditions that allow interaction of theantibody molecule and the polypeptide to occur, and (ii) detectingformation of a complex between the antibody molecule and the sample orthe subject (and optionally, the reference sample or subject).

In some aspects, this disclosure provides a formulation as describedherein that includes an anti-dengue antibody molecule comprising a VHregion that has a deletion of position 26 relative to the VH of antibodyA11. For instance, the anti-dengue antibody molecule of claim may have aVH region with between about 1-30, 5-30, 10-30, 15-30, or 20-25mutations relative to a VH of antibody A11.

In some aspects, this disclosure provides a formulation as describedherein that includes an antibody molecule capable of binding to denguevirus, which comprises a VH CDR1 having the sequence of SEQ ID NO: 3, oran amino acid sequence having no more than 1, 2, 3, 4, 5, 10, or 15mutations relative to SEQ ID NO: 3. In some embodiments, the mutationsare substitutions, e.g., conservative substitutions.

In certain aspects, this disclosure provides a formulation as describedherein that includes an antibody molecule capable of binding to denguevirus, which comprises a VH FW1 having the sequenceQVQLVQSGAEVKKPGASVKVSCKAGFNIK (SEQ ID NO: 11), or an amino acid sequencehaving no more than 1, 2, 3, 4, 5, 10, or 15 mutations relative to SEQID NO: 11. In some embodiments, the mutations are independently selectedfrom deletions and substitutions, e.g., conservative substitutions. Anantibody molecule of this paragraph may also have the features describedthroughout this application, e.g., in the previous paragraph.

In some aspects, this disclosure provides a formulation as describedherein that includes antibody molecules capable of binding to denguevirus, which comprises a VH CDR2 having the sequence of SEQ ID NO: 4, oran amino acid sequence having no more than 1, 2, 3, 4, 5, 10, or 15mutations relative to SEQ ID NO: 4. In some embodiments, the mutationsare substitutions, e.g., conservative substitutions. An antibodymolecule of this paragraph may also have the features describedthroughout this application, e.g., in the previous two paragraphs.

In certain aspects, this disclosure provides a formulation as describedherein that includes an antibody molecule capable of binding to denguevirus, which comprises a VH FW2 having the sequence WVRQAPGQGLEWMG (SEQID NO: 84) or WVRQAPEQGLEWMG (SEQ ID NO: 85), or an amino acid sequencehaving no more than 1, 2, 3, 4, 5, or 10 mutations relative to SEQ IDNO: 84 or SEQ ID NO: 85. In some embodiments, the mutations areindependently selected from deletions and substitutions, e.g.,conservative substitutions. An antibody molecule of this paragraph mayalso have the features described throughout this application, e.g., inthe previous paragraph.

In some embodiments, the antibody molecule is capable of binding toEDIII of two or more, e.g., three or four, dengue virus serotypes, e.g.,selected from DV-1, DV-2, DV-3, and DV-4, with a dissociation constant(K_(D)) of less than about 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4, 3,2, 1, 0.5, 0.2, 0.1, 0.05, or 0.01 nM for each of said two or moreserotypes.

In some embodiments, the antibody molecule has a variable region that isidentical in sequence, or which differs by 1, 2, 3, or 4 amino acidsfrom a variable region described herein (e.g., an FR region disclosedherein).

In some embodiments, the anti-dengue antibody molecule is a fullantibody or fragment thereof (e.g., a Fab, F(ab′)2, Fv, or a singlechain Fv fragment (scFv)). In certain embodiments, the anti-dengueantibody molecule is a monoclonal antibody or an antibody with singlespecificity. The anti-dengue antibody molecule can also be a humanized,chimeric, camelid, shark, or in vitro-generated antibody molecule. Insome embodiments, the anti-dengue antibody molecule thereof is ahumanized antibody molecule. The heavy and light chains of theanti-dengue antibody molecule can be full-length (e.g., an antibody caninclude at least one or at least two complete heavy chains, and at leastone or at least two complete light chains) or can include anantigen-binding fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fvfragment, a single domain antibody, a diabody (dAb), a bivalent orbispecific antibody or fragment thereof, a single domain variantthereof, or a camelid antibody).

In certain embodiments, the anti-dengue antibody molecule has a heavychain constant region (Fc) chosen from, e.g., the heavy chain constantregions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE;particularly, chosen from, e.g., the heavy chain constant regions ofIgG1, IgG2, IgG3, and IgG4, more particularly, the heavy chain constantregion of IgG1 or IgG2 (e.g., human IgG1 or IgG2). In some embodiments,the heavy chain constant region is human IgG1. In some embodiments, theanti-dengue antibody molecule has a light chain constant region chosenfrom, e.g., the light chain constant regions of kappa or lambda, in someembodiments kappa (e.g., human kappa). In some embodiments, the constantregion is altered, e.g., mutated, to modify the properties of theanti-dengue antibody molecule (e.g., to increase or decrease one or moreof: Fc receptor binding, antibody glycosylation, the number of cysteineresidues, effector cell function, or complement function). For example,the constant region may be mutated at positions 234 (e.g., L to A), 235(e.g., L to A), 296 (e.g., M to Y), 297 (e.g., N to A or G or Q), 298(e.g., S to T), 300 (e.g., T to E), 477 (e.g., H to K) and 478 (e.g., Nto F) to alter Fc receptor binding.

In some embodiments, the antibody molecule is a humanized antibodymolecule. In some embodiments, the antibody molecule is isolated orrecombinant. In some embodiments, the anti-dengue antibody moleculescomprise combinations of human or humanized framework regions with CDRs(complementarity determining regions).

The present disclosure also provides nucleic acids comprising nucleotidesequences that encode heavy and light chain variable regions and CDRs ofthe anti-dengue antibody molecules, as described herein. For example,the disclosure provides a first and second nucleic acid encoding heavyand light chain variable regions, respectively, of an anti-dengueantibody molecule according to Table 1, e.g., D88, A48, F38, F108, orC88, or a sequence substantially identical thereto. For example, thenucleic acid can comprise a nucleotide sequence encoding an anti-dengueantibody molecule according to Table 1, e.g., D88, A48, F38, F108, orC88, or a sequence substantially identical to that nucleotide sequence(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, or which differs by no more than 3, 6, 15, 30, or 45nucleotides from the aforementioned nucleotide sequence). In certainembodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, or three CDRs from a heavy chain variableregion having an amino acid sequence as set forth in Table 2, or asequence substantially homologous thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or having one,two, three or more substitutions, insertions or deletions, e.g.,conserved substitutions). In certain embodiments, the nucleic acid cancomprise a nucleotide sequence encoding at least one, two, or three CDRsfrom a light chain variable region having an amino acid sequence as setforth in Table 2, or a sequence substantially homologous thereto (e.g.,a sequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or having one, two, three or more substitutions, insertions ordeletions, e.g., conserved substitutions). In some embodiments, thenucleic acid can comprise a nucleotide sequence encoding at least one,two, three, four, five, or six CDRs from heavy and light chain variableregions having an amino acid sequence as set forth in Table 2, or asequence substantially homologous thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or having one,two, three or more mutations (e.g., substitutions, insertions ordeletions, e.g., conserved substitutions). In some embodiments, anucleic acid having a structural feature discussed in this paragraphencodes an antibody molecule or portion thereof having one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

This disclosure also provides nucleic acid sequences, e.g., a nucleicacid that hybridizes to a nucleic acid sequence that encodes an antibodyof Table 1, e.g., D88, A48, F38, F108, or C88, and its complement, e.g.,under low stringency, medium stringency, or high stringency, or otherhybridization condition described herein. This application alsodiscloses nucleic acid sequences of Table 4, and their complements,e.g., under low stringency, medium stringency, or high stringency, orother hybridization condition described herein. In some embodiments, thenucleic acid is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical to a sequence of Table 4, its complement, or a portionof any of the aforementioned sequences. In some embodiments, a nucleicacid having a structural feature discussed in this paragraph encodes anantibody molecule or portion thereof having one or more advantageousproperties such as an improved (e.g., relative to A11) affinity for orneutralization activity towards dengue virus, e.g., DV-4. In someembodiments, the advantageous property is a property of List 1, e.g.,one or more (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all) ofproperties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s),(t), (u), (v), (w), or (x).

In certain aspects, this disclosure features host cells and vectorscontaining the nucleic acids described herein. The nucleic acids may bepresent in a single vector or separate vectors present in the same hostcell or separate host cell. The host cell can be a eukaryotic cell,e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryoticcell, e.g., E. coli. For example, the mammalian cell can be a culturedcell or a cell line. Exemplary mammalian cells include human cells e.g.,HEK293 cells, lymphocytic cell lines (e.g., NSO), Chinese hamster ovarycells (CHO), COS cells, oocyte cells, and cells from a transgenicanimal.

In some aspects, the present disclosure provides a method of providingan antibody molecule described herein, e.g., in a formulation describedherein. The method may include: providing an antibody molecule thatspecifically binds to an EDIII antigen; making one or more mutations tothe antibody (e.g., to the constant region, framework, and/or CDRs) andevaluating if the antibody molecule specifically binds to the EDIIIantigen, or evaluating efficacy of the antibody molecule in inhibitingdengue virus function. The method can further include purifying theantibody molecule. For example, the antibody molecule can be purified byone or more chromatography steps comprising, e.g., affinitychromatography, e.g., Protein A chromatography. The method can furtherinclude administering the antibody molecule to a subject, e.g., a humanor non-human animal.

In certain aspects, the disclosure provides, formulations orcompositions, e.g., pharmaceutical compositions, which include apharmaceutically acceptable carrier, excipient or stabilizer, and atleast one of the anti-dengue antibody molecules described herein. Insome embodiments, the antibody molecule is conjugated to a label or atherapeutic agent. In some embodiments, the formulations, e.g.,compositions, e.g., the pharmaceutical compositions, comprise acombination of the antibody molecule and a second agent, e.g., atherapeutic agent, or two or more of the aforesaid antibody molecules,as further described herein.

The antibody molecules disclosed herein can inhibit one or moreactivities of dengue virus. For instance, the antibody molecules maydisrupt the native structure of the E protein on the surface of thevirion, e.g., causing inactivation of the virus. As a result, theantibody molecule may neutralize the virus, inhibit its ability to entera host cell, or reduce viral stability. In some embodiments, an antibodymolecule neutralizes dengue virus (e.g., in a focus reductionneutralization test or a related test for evaluating neutralization ofviral activity) with an EC50 or FRNT50 of less than or equal to 1400,1000, 800, 600, 550, 500, 450, 400, 350, 300, 350, 200, 150, 100, 50, or25 ng/ml. In some embodiments, the antibody neutralizes dengue virus(e.g., in a focus reduction neutralization test or a related test forevaluating neutralization of viral activity) with an IC50 of less thanor equal to 20, 17.6, 15, 10, 5, 4, 2, 1.4, 1, or 0.50 μg/mL. In someembodiments, neutralization of DV-4 is tested, and in some embodiments,neutralization of one of DV-1, DV-2, or DV-3 is tested.

The subject can be a mammal, e.g., a monkey, a primate, preferably ahigher primate, e.g., a human (e.g., a patient having, or at risk ofhaving, dengue virus).

This disclosure also provides a method of treating dengue virus in asubject, comprising administering to the subject an anti-dengue antibodymolecule described herein, e.g., a therapeutically effective amount ofan anti-dengue antibody molecule, or an antigen-binding portion thereof.In some embodiments, the anti-dengue antibody molecule is administeredto a patient suffering from dengue virus, resulting in the reduction ofviral load and/or the reduction of at least one symptom, e.g., selectedfrom sudden-onset fever, headache, muscle and joint pains, weight loss,central nervous system penetration, and/or rash. In some embodiments,the anti-dengue antibody molecule is administered to a patient at riskof being infected from dengue virus, resulting in the reduction of therisk of becoming infected or reduction of the severity of the infectionif infection does occur.

The anti-dengue antibody molecule can be administered to the subjectsystemically (e.g., orally, parenterally, subcutaneously, intravenously,rectally, intramuscularly, intraperitoneally, intranasally,transdermally, or by inhalation or intracavitary installation),topically, or by application to mucous membranes, such as the nose,throat and bronchial tubes.

The methods and formulations, e.g., compositions, described herein canbe used in combination with other therapeutic modalities. In someembodiments, the methods of described herein include administering tothe subject an anti-dengue antibody molecule as described herein, incombination with a second treatment or prophylactic for dengue virus, inan amount effective to treat or prevent said disorder. The antibodymolecule and the second agent can be administered simultaneously orsequentially.

Any combination and sequence of the anti-dengue antibody molecules andother therapeutic modalities can be used. The anti-dengue antibodymolecule and/or other therapeutic modalities can be administered duringperiods of active infection, or during a period of remission or lessactive disease. The anti-dengue antibody molecule and other therapeuticmodalities can be administered before treatment, concurrently withtreatment, post-treatment, or during remission of the infection.

In some aspects, the present disclosure provides methods for detectingthe presence of dengue virus in a sample, e.g., in vitro or in vivo(e.g., a biological sample, e.g., blood or serum). The methods hereincan be used to evaluate (e.g., monitor treatment or progression of,diagnose and/or stage a disorder described herein, e.g., dengue virus,in a subject). The method may include: (i) contacting the sample with(and optionally, a reference, e.g., a control sample), or administeringto the subject, an anti-dengue antibody molecule as described herein,under conditions that allow interaction to occur, and (ii) detectingwhether there is formation of a complex between the antibody moleculeand the sample (and optionally, the reference, e.g., control, sample).Formation of the complex is indicative of the presence of dengue virus,and can indicate the suitability or need for a treatment describedherein. The method can involve, e.g., an immunohistochemistry,immunocytochemistry, FACS, antibody molecule-complexed magnetic beads,ELISA assays, or PCR-techniques (e.g., RT-PCR).

Typically, the anti-dengue antibody molecule used in the in vivo and invitro diagnostic methods is directly or indirectly labeled with adetectable substance to facilitate detection of the bound or unboundbinding agent. Suitable detectable substances include variousbiologically active enzymes, prosthetic groups, fluorescent materials,luminescent materials, paramagnetic (e.g., nuclear magnetic resonanceactive) materials, and radioactive materials.

In some aspects, the present disclosure provides diagnostic ortherapeutic kits that include the anti-dengue antibody moleculesdescribed herein and instructions for use.

The disclosure contemplates all combinations of any one or more of theforegoing aspects and/or embodiments, as well as combinations with anyone or more of the embodiments set forth in the detailed description andexamples.

Other features, objects, and advantages of the formulations, e.g.,compositions, and methods herein will be apparent from the descriptionand drawings, and from the claims.

Figures and Tables are provided herewith.

BRIEF DESCRIPTION OF THE DRAWINGS

Each of the Figures is described herein in more detail.

FIGS. 1A-1I show the amino acid and nucleotide sequences of severalanti-dengue EDIII antibodies. Kabat CDRs are underlined, and certainresidues of interest are boxed (shown with a gray background in thepriority documents).

FIGS. 2A-2B show the osmolality (FIG. 2A) and pH (FIG. 2B) of theformulations in Table 7.

FIG. 3 shows visual appearance data for the formulations in Table 7.

FIG. 4 shows gel permeation high performance liquid chromatography (GPHPLC) data for the formulations in Table 7.

FIG. 5 shows the relative percentage of IgG band area by reduced SDSPAGE for the formulations in Table 7.

FIG. 6 shows the number of bands detected visually by non-reduced SDSPAGE for the formulations in Table 7.

FIG. 7 shows isoelectric focusing data for the formulations in Table 7at 5° C.

FIG. 8 shows isoelectric focusing data for the formulations in Table 7at 40° C.

FIG. 9 shows the absorbance at 340 nm and at 620 nm for the formulationsin Table 7.

FIG. 10 shows dynamic light scattering data for the formulations inTable 7.

FIG. 11 shows differential scanning calorimetry data for theformulations in Table 7.

FIG. 12 shows sub-visible particle characterization by microflow imagingfor the formulations in Table 7.

BRIEF DESCRIPTION OF THE TABLES

Each of the Tables is described herein in more detail.

Table 1 summarizes the sequences of exemplary anti-dengue antibodies.

Table 2 depicts the amino acid sequences of the heavy chain variabledomain and light chain variable domain sequences of Table 1. Kabat CDRsare underlined, Chothia CDRs are italicized, and certain residues ofinterest are shown with a gray background.

Table 3 depicts the amino acid sequences of the CDRs of Table 1.

Table 4 summarizes the nucleic acid sequences encoding the antibodies ofTable 1.

Table 5 depicts the nucleic acid sequences summarized in Table 4.

Table 6 depicts additional amino acid sequences described throughout theapplication.

Table 7 describes 21 formulations analyzed in the study of Example 1.

Table 8 describes the timepoint schedule used in the study of Example 1.

Table 9 summarizes the results of the study of Example 1.

DETAILED DESCRIPTION

Disclosed herein are formulations that include antibody molecules thatbind to dengue virus epitopes, e.g., EDIII, with high affinity andspecificity. The formulations disclosed herein have superior propertiescompared to other antibody formulations, including, but not limited to,increased stability, decreased degradation, decreased aggregation, orincreased shelf life. Advantageously, the formulations disclosed hereininclude several of the antibody molecules herein bind with high affinityto EDIII of dengue virus serotypes DV-1, DV-2, DV-3, and DV-4. Nucleicacid molecules encoding the antibody molecules, expression vectors, hostcells and methods for making the antibody molecules are also provided.The anti-dengue antibody molecules disclosed herein can be used (aloneor in combination with other agents or therapeutic modalities) to treat,prevent and/or diagnose dengue virus, e.g., DV-1, DV-2, DV-3, or DV-4.As used herein, DV-1, DV-2, DV-3, and DV-4 are sometimes referred to asDENV-1, DENV-2, DENV-3, and DENV-4, respectively.

Definitions

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

The compositions and methods disclosed herein encompass polypeptides andnucleic acids having the sequences specified, or sequences substantiallyidentical or similar thereto, e.g., sequences at least 85%, 90%, 95%identical or higher to the sequence specified. In the context of anamino acid sequence, the term “substantially identical” is used hereinto refer to a first amino acid that contains a sufficient or minimumnumber of amino acid residues that are i) identical to, or ii)conservative substitutions of aligned amino acid residues in a secondamino acid sequence such that the first and second amino acid sequencescan have a common structural domain and/or common functional activity.For example, amino acid sequences that contain a common structuraldomain having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity to a reference sequence, e.g., a sequenceprovided herein.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence,e.g., a sequence provided herein.

The term “functional variant” refers polypeptides that have asubstantially identical amino acid sequence to the naturally-occurringsequence, or are encoded by a substantially identical nucleotidesequence, and are capable of having one or more activities of thenaturally-occurring sequence.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, e.g., at least 40%, 50%, 60%, e.g.,at least 70%, 80%, 90%, 100% of the length of the reference sequence.The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In some embodiments, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch ((1970) J.Mol. Biol. 48:444-453) algorithm which has been incorporated into theGAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In certain embodiments, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. One suitable set ofparameters (and the one that should be used unless otherwise specified)are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extendpenalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid as described herein. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules described herein. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used. Seewww.ncbi.nlm.nih.gov.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) aresuitable conditions and the ones that should be used unless otherwisespecified.

It is understood that the molecules described herein may have additionalconservative or non-essential amino acid substitutions, which do nothave a substantial effect on their functions.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

The terms “polypeptide,” “peptide” and “protein” (if single chain) areused interchangeably herein.

The terms “nucleic acid,” “nucleic acid sequence,” “nucleotidesequence,” or “polynucleotide sequence,” and “polynucleotide” are usedinterchangeably.

The term “isolated,” as used herein, refers to material that is removedfrom its original or native environment (e.g., the natural environmentif it is naturally occurring). For example, a naturally-occurringpolynucleotide or polypeptide present in a living animal is notisolated, but the same polynucleotide or polypeptide, separated by humanintervention from some or all of the co-existing materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of the environment in which it is found in nature.

As used herein, the term “treat”, e.g., a dengue virus infection, meansthat a subject (e.g., a human) who has been infected with a virus andexperiences symptoms of the virus, will, in embodiments, suffer lesssevere symptoms and/or will recover faster when the antibody molecule isadministered than if the antibody were never administered. Inembodiments, when an infection is treated, an assay to detect virus inthe subject will detect less virus after effective treatment for theinfection. For example, a diagnostic assay using an antibody molecule,such as an antibody molecule described herein, will detect less or novirus in a biological sample of a patient after administration of anantibody molecule for the effective treatment of the infection. Otherassays, such as PCR (e.g., qPCR) can also be used to monitor treatmentin a patient, to detect the presence, e.g., decreased presence (orabsence) after treatment of viral infection in the patient. Treatmentcan, e.g., partially or completely alleviate, ameliorate, relieve,inhibit, reduce the severity of, and/or reduce incidence and optionally,delay onset of, one or more manifestations of the effects or symptoms,features, and/or causes of a particular disease, disorder, and/orcondition (e.g., dengue virus). In embodiments treatment is of a subjectwho does not exhibit certain signs of the relevant disease, disorderand/or condition and/or of a subject who exhibits only early signs ofthe disease, disorder, and/or condition. In some embodiments, treatmentis of a subject who exhibits one or more established signs of therelevant disease, disorder and/or condition. In some embodiments,treatment is of a subject diagnosed as suffering from dengue virus.

As used herein, the term “prevent”, e.g., a dengue virus infection,means that a subject (e.g., a human) is less likely to be infected by avirus (e.g., dengue virus) if the subject receives the antibody prior to(e.g., 1 day, 2 days, 1 week, 2 weeks, 3 weeks, or 1 month of more)being exposed to the virus.

As used herein, the terms “framework,” “FW” and “FR” are usedinterchangeably and have identical meaning in this document and itspriority documents.

Various aspects of the compositions and methods herein are described infurther detail below. Additional definitions are set out throughout thespecification.

Anti-Dengue Antibody Molecules

Exemplary sequences of anti-dengue antibodies are described in Tables1-4 below.

TABLE 1 Summary of the amino acid sequences of exemplary anti-dengueantibodies Antibody designation SEQ ID NO Description D88 1 VH aminoacid sequence 2 VL amino acid sequence 3 VH CDR1 amino acid sequence,Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acidsequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 aminoacid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 9 VH CDR1amino acid sequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acid sequence,Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 amino acidsequence, Chothia 11 VH FW1 amino acid sequence, Kabat 85 VH FW2 aminoacid sequence, Kabat F38 80 VH amino acid sequence 2 VL amino acidsequence 3 VH CDR1 amino acid sequence, Kabat 4 VH CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 9 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia 11 VH FW1 amino acidsequence, Kabat 84 VH FW2 amino acid sequence, Kabat A48 16 VH aminoacid sequence 2 VL amino acid sequence 14 VH CDR1 amino acid sequence,Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acidsequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 aminoacid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 15 VH CDR1amino acid sequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acid sequence,Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 amino acidsequence, Chothia C88 17 VH amino acid sequence 2 VL amino acid sequence3 VH CDR1 amino acid sequence, Kabat 4 VH CDR2 amino acid sequence,Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 amino acidsequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3 aminoacid sequence, Kabat 9 VH CDR1 amino acid sequence, Chothia 10 VH CDR2amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VLCDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence, Chothia8 VL CDR3 amino acid sequence, Chothia 85 VH FW2 amino acid sequence,Kabat F108 81 VH amino acid sequence 2 VL amino acid sequence 3 VH CDR1amino acid sequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VHCDR3 amino acid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7VL CDR2 amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat9 VH CDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia 84 VH FW2 amino acid sequence, Kabat B48 18 VHamino acid sequence 2 VL amino acid sequence 14 VH CDR1 amino acidsequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 aminoacid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 15 VHCDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia A68 19 VH amino acid sequence 2 VL amino acidsequence 14 VH CDR1 amino acid sequence, Kabat 4 VH CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 15 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia A100 20 VH amino acidsequence 2 VL amino acid sequence 14 VH CDR1 amino acid sequence, Kabat4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acid sequence,Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 amino acidsequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 15 VH CDR1 aminoacid sequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5 VH CDR3amino acid sequence, Chothia 6 VL CDR1 amino acid sequence, Chothia 7 VLCDR2 amino acid sequence, Chothia 8 VL CDR3 amino acid sequence, ChothiaC58 21 VH amino acid sequence 2 VL amino acid sequence 14 VH CDR1 aminoacid sequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3amino acid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VLCDR2 amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 22VH CDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia C78 23 VH amino acid sequence 2 VL amino acidsequence 3 VH CDR1 amino acid sequence, Kabat 4 VH CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 24 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia C68 25 VH amino acidsequence 2 VL amino acid sequence 3 VH CDR1 amino acid sequence, Kabat 4VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acid sequence, Kabat6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 amino acid sequence,Kabat 8 VL CDR3 amino acid sequence, Kabat 26 VH CDR1 amino acidsequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5 VH CDR3amino acid sequence, Chothia 6 VL CDR1 amino acid sequence, Chothia 7 VLCDR2 amino acid sequence, Chothia 8 VL CDR3 amino acid sequence, ChothiaD98 27 VH amino acid sequence 2 VL amino acid sequence 14 VH CDR1 aminoacid sequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3amino acid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VLCDR2 amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 28VH CDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia D188 29 VH amino acid sequence 2 VL amino acidsequence 3 VH CDR1 amino acid sequence, Kabat 4 VH CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 30 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia C128 31 VH amino acidsequence 2 VL amino acid sequence 3 VH CDR1 amino acid sequence, Kabat 4VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acid sequence, Kabat6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 amino acid sequence,Kabat 8 VL CDR3 amino acid sequence, Kabat 9 VH CDR1 amino acidsequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5 VH CDR3amino acid sequence, Chothia 6 VL CDR1 amino acid sequence, Chothia 7 VLCDR2 amino acid sequence, Chothia 8 VL CDR3 amino acid sequence, ChothiaC98 32 VH amino acid sequence 2 VL amino acid sequence 3 VH CDR1 aminoacid sequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3amino acid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VLCDR2 amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 9VH CDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia A11 33 VH amino acid sequence 34 VL amino acidsequence 14 VH CDR1 amino acid sequence, Kabat 35 VH CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 15 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia B11 36 VH amino acidsequence 34 VL amino acid sequence 14 VH CDR1 amino acid sequence, Kabat21 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acid sequence,Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 amino acidsequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 15 VH CDR1 aminoacid sequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5 VH CDR3amino acid sequence, Chothia 6 VL CDR1 amino acid sequence, Chothia 7 VLCDR2 amino acid sequence, Chothia 8 VL CDR3 amino acid sequence, Chothia

A11 and B11 are mouse antibodies, and the other antibodies of Table 1are humanized antibodies.

TABLE 2Depiction of the amino acid sequences of the heavy chain variable domain and light chainvariable domain sequences of Table 1. SEQ Description ID NO SequenceD88 VH  1

D88 VL  2

GTKLEIK F38 VH 80

A48 VH 16

C88 VH 17

F108 VH 81

B48 VH 18

A68 VH 19

A100 VH 20

C58 VH 21

C78 VH 23

C68 VH 25

D98 VH 27

D188 VH 29

C128 VH 31

C98 VH 32

A11 VH 33

A11 VL 34

GTKLEIK B11 VH 36

CDRs, defined according to the Kabat system, are underlined and bold,while CDRs defined according to the Chothia system are italicized.Certain residues of interest are shown with a gray background. Deletionsare indicated with a caret symbol (+).

Throughout this application, reference is made to amino acid positionsbased on the variable region of mouse antibody A11. The variable regionof mouse antibody B11 has a deletion at position 26 relative to A11. Thehuman variable region sequences in Table 1 have a deletion of theglutamic acid sequence at position 6 of A11. Consequently, sequencesthat carry a deletion relative to A11 use a numbering system that isoffset. For example, A48 heavy chain has a deletion of the glutamic acidsequence at position 6 relative to A11. As a result, position 26 (aserine) of A48 VH is actually the twenty-fifth amino acid of the A48 VHsequence (SEQ ID NO: 16). As another example, D88 heavy chain has adeletion of the glutamic acid sequence at position 6 of A11 and adeletion of the serine at position 26 relative to A11. As a consequence,position 33 (a valine) of D88 VH is actually the thirty-first amino acidof the D88 VH sequence (SEQ ID NO: 1).

Some structural features of the antibodies can be noted based on the VHand VL sequences in Table 2. B11 has a deletion at position 26 relativeto the A11 VH region. D88 is a humanized antibody that has a deletion atposition 26 and a T33V mutation relative to the A48 VH region (remainingconsistent with A11 numbering). F38 is a humanized antibody that has adeletion at position 26 and T33V and E43G mutations relative to the A48VH region. A48 is a humanized antibody with the same CDRs as A11. C88 isa humanized antibody that has a T33V mutation relative to the A48 VHregion. F108 is a humanized antibody that has T33V and E43G mutationsrelative to the A48 VH region. B48 is a humanized antibody that has adeletion at position 26 relative to the A48 VH region. A68 is ahumanized antibody that has an A98V mutation relative to the A48 VHregion. A100 is a humanized antibody that has an A98S mutation relativeto the A48 VH region. C58 is a humanized antibody that has G27Y and F28Wmutations relative to the A48 VH region. C78 is a humanized antibodythat has K31Q and T33V mutations relative to the A48 VH region. C68 is ahumanized antibody that has K31S and T33V mutations relative to the A48VH region. D98 is a humanized antibody that has a G27A mutation relativeto the A48 VH region.

Other variations of the antibodies of Tables 1 and 2 are envisioned. Forinstance, this application provides antibody B48+A98V, which has an A98Vmutation relative to B48; A48+V2L, which has a V2L mutation relative toA48; A48+InsE6, which has an InsE6 mutation relative to A48; B48+V2L,which has a V2L mutation relative to B48; B48+InsE6, which has an InsE6mutation relative to B48; D118, which has F28W and T33V mutationsrelative to A48; D128, which has G27A, F28W, and T33V mutations relativeto A48; D138, which has G27Y, F28A, and T33V mutations relative to A48;D148, which has G27Y and T33V mutations relative to A48; D158, which hasG27Y, F28G, and T33V mutations relative to A48; D168, which has F28Y andT33V mutations relative to A48; C98, which has T33V and A98V mutationsrelative to A48; C128, which has T33V and A98S mutations relative toA48; D178, which has De126, T33V, and A98V mutations relative to A48;and D188, which has De126, T33V, and A98S mutations relative to A48.

TABLE 3 Depiction of the amino acid sequences of the CDRs of Table 1 SEQID NO Sequence  3 DVYMS  4 RIDPENGDTKYDPKLQG  5 GWEGFAY  6RASENVDKYGNSFMH  7 RASELQW  8 QRSNEVPWT  9 GFNIKDV 10 DPENGD 14 DTYMS 15GFNIKDT 22 YWNIKDT 24 GFNIQDV 26 GFNISDV 28 AFNIKDT 30 AFNIKDV 35RIDPENGDTKYDPKFQG

TABLE 4 Summary of the nucleic acid sequences encoding the antibodies ofTable 1 Antibody designation SEQ ID NO Description D88 37 VH nucleicacid sequence 38 VL nucleic acid sequence F38 82 VH nucleic acidsequence 38 VL nucleic acid sequence A48 39 VH nucleic acid sequence 38VL nucleic acid sequence C88 40 VH nucleic acid sequence 38 VL nucleicacid sequence F108 83 VH nucleic acid sequence 38 VL nucleic acidsequence B48 41 VH nucleic acid sequence 38 VL nucleic acid sequence A6842 VH nucleic acid sequence 38 VL nucleic acid sequence A100 86 VHnucleic acid sequence 38 VL nucleic acid sequence C58 43 VH nucleic acidsequence 38 VL nucleic acid sequence C78 44 VH nucleic acid sequence 38VL nucleic acid sequence C68 45 VH nucleic acid sequence 38 VL nucleicacid sequence D98 46 VH nucleic acid sequence 38 VL nucleic acidsequence D188 87 VH nucleic acid sequence 38 VL nucleic acid sequenceC128 88 VH nucleic acid sequence 38 VL nucleic acid sequence C98 89 VHnucleic acid sequence 38 VL nucleic acid sequence A11 47 VH nucleic acidsequence 48 VL nucleic acid sequence B11 49 VH nucleic acid sequence 48VL nucleic acid sequence

TABLE 5 Nucleic acid sequences of Table 4 SEQ ID NO Sequence 37CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 38GATATTGTCATGACCCAAAGCCCAGCCTCCCTCGCCGTGTCTCTCGGAGAAAGAGCAACTATCTCGTGCCGGGCTTCGGAGAATGTGGACAAGTACGGCAACTCCTTCATGCACTGGTACCAGCAGAAACCGGGACAGCCGCCTAAACTGTTGATCTACCGGGCGTCAGAACTGCAATGGGGAGTGCCTGACAGGTTTTCGGGTTCGGGATCCGGCACGGATTTCACCCTCACTATCTCCAGCCTGCAAGCAGAGGACGTTGCGGTGTACTACTGTCAGCGCTCAAACGAGGTCCCATGGACTTTTGGACAAGGGACCAAGCTGGAAATCAAG 82CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGGGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 39CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 40CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 83CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGGGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 41CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCGGCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 42CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGTCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 86CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTTCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 43CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGTACTGGAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 44CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCCAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 45CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCTCGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 46CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGCCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 87CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGCCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 88CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTAGCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 89CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 47CAAGTCAAACTGCTGGAACAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCGTCGGTGCGGCTTTCGTGTACCGCCTCCGGCTTTAACATCAAGGACACCTACATGTCGTGGGTGAAGCAGAGGCCCGAGCAGGGGCTCGAATGGATTGGCCGCATCGACCCGGAAAATGGTGATACCAAATACGACCCAAAGTTCCAGGGAAAGGCCACTATCACTGCAGATACTTCAAGCAACACCGCCTACCTCCACCTGTCCTCGCTCACTTCCGGAGATACCGCGGTCTACTATTGCTCAAGAGGATGGGAAGGCTTCGCGTACTGGGGTCAAGGAACGTTGGTGACCGTCAGCGCC 48GAATTGGTCATGACTCAGACGCCAGCTTCGCTGGCCGTGTCACTGGGACAGAGGGCCACTATCAGCTGCAGAGCATCGGAGAATGTGGATAAGTACGGGAACAGCTTCATGCACTGGTATCAACAGAAAGCTGGTCAACCTCCGAAGCTGCTTATCTACCGGGCGTCGGAACTCCAATGGGGCATTCCAGCACGGTTCAGCGGGTCGGGCTCCAGAACTGACTTCACCCTCACCATCAATCCCGTGGAGGCCGATGACGTGGCGACCTACTTTTGTCAGCGCTCCAACGAGGTCCCGTGGACTTTCGGAGGAGGAACCAAGCTGGAAATCAAG 49CAAGTCAAACTGCTGGAACAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCGTCGGTGCGGCTTTCGTGTACCGCCGGCTTTAACATCAAGGACACCTACATGTCGTGGGTGAAGCAGAGGCCCGAGCAGGGGCTCGAATGGATTGGCCGCATCGACCCGGAAAATGGTGATACCAAATACGACCCAAAGTTCCAGGGAAAGGCCACTATCACTGCAGATACTTCAAGCAACACCGCCTACCTCCACCTGTCCTCGCTCACTTCCGGAGATACCGCGGTCTACTATTGCTCAAGAGGATGGGAAGGCTTCGCGTACTGGGGTCAAGGAACGTTGGTGACCGTCAGCGCC

TABLE 6 Additional amino acid sequences SEQ ID Description NO SequenceFW1 region of SEQ 11 QVQLVQSGAEVKKPGASVKVSCKA{circumflex over ( )}GFNIKID NO: 1 FW2 region of SEQ 84 WVRQAPGQGLEWMG ID NO: 80 FW2 region of SEQ85 WVRQAPEQGLEWMG ID NO: 1 EDIII-DV1 50MTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGATQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVG AGEKALKLSWFKKGSSIGKEDIII-DV2 51 MQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIG VEPGQLKLNWFKKGSSLEEDIII-DV3 52 MKLKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIG IGDKALKINWYRKGSSIGKEDIII-DV4 53 MRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIG VGNSALTLHWFRKGSSIGKEDIII sequences for testing breadth of binding ED3-DV1/Viet08 54MTLKGMSYVMCTGSFKLEKELAETQHGTVLVQIKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVIG AGEKALKLSWFKKGSSIGK ED3-55 MTLKGISYVMCTGPFKLEKEVAETQHGTVLVQVKYEGTDAPCKIP DV1/Malaysia05FSSQDEKGVTQNGRLVTANPIVTDKEKPVNIEAEPPFGESYIVVG AGEKALKLSWFKKGSSIGKED3-DV1/Mexico07 56 MTLKGTSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNGRLITANPIVTDKEKPVNIETEPPFGESYIVVG AGEKALKLSWFKKGSSIGKED3-DV2/Sing08 57 MQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIG VEPGQLKLSWFKKGSSIGQ ED3-58 MQLKGMSYSMCTGKFKIVKEIAETQHGTIVIRIQYEGDGSPCKIP DV2/Venezuela07FEITDLEKRHVLGRLITVNPIVIEKDSPVNIEAEPPFGDSYIIIG VEPGQLKLNWFKKGSSIGQED3-DV2/Peru95 59 MQLKGMSYSMCTGKFKIVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIG VEPGQLKLDWFKKGSSIGQED3-DV2/Viet07 60 MQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRYVLGRLITVNPIVTEKDSPINIEAEPPFGDSYIIIG VEPGQLKLNWFKKGSSIGQ ED3-61 MELKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIP DV3/Cambodia08FSTEDGQGKAHSGRLITANPVVTKKEEPVNIEAEPPFGESNIVIG IGDKALKINWYKKGSSIGKED3-DV3/Sing09 62 MELKGMSYAMCQNAFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIG IGDKALKINWYKKGSSIGK ED3-63 MELKGMSYAMCTNTFVLKKEVSETQHGTILIKVEYKGEDVPCKIP DV3/Nicaragua10FSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIG IGDNALKINWYKKGSSIGK ED3-64 MELKGMSYAMCSGTFVLKKEVSETQHGTILIKIEYKGEDAPCKIP DV3/PuertoRico77FSTEDAQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIG TGDKALRINWYKKGSSIGK ED3-65 MRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVP DV4/Venezuela08IEIRDVNKEKVVGRVISATPLAENTNSVTNIELEPPFGDSYIVIG VGNSALTLHWFRKGSSIGKED3-DV4/Sing10 66 MRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPFAENTNSVTNIELEPPFGDSYIVIG VGDSALTLHWFRKGSSIGKED3-DV4/NewCal09 67 MRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKIPIEIRDVNKEKVVGRIISSTPFAENTNSVINIELEPPFGDSYIVIG VGDSALTLHWFRKGSSIGKED3-DV4/Brazil11 68 MRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKIKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPFAENTNSVTNIELEPPFGDSYIVIG VGDSALTLHWFRKGSSIGKED3-DV4/Thai97 69 MRIKGMSYTMCSGKFSIDREMAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRIISSTPFAESTNSVTNIELEPPFGDSYIVIG VGDSALTLHWFRKGSSIGK ED3-70 MRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVP DV4/H241/Phil56IEIRDVNKEKVVGRIISSTPFAEYTNSVTNIELEPPFGDSYIVIG VGDSALTLHWFRKGSSIGKHuman germline sequences, heavy chain Human germline = 71QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGL VH1-69, JH4EWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSED TAVYYCARYFDYWGQGTLVTVSSHuman germline = 72 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLVH1-18, JH6 EWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARYMDVWGKGTTVTVSS Human germline = 73QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGL VH1-18, JH4EWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCARYMDVWGQGTLVTVSSHuman germline = 74 EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWISWVRQMPGKGLVH5-a*04, JH4 EWMGRIDPSDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARYMDVWGQGTLVTVSS Human germline = 75QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYYMHWVRQAPGQGL VH1-46, JH4EWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARYFDYWGQGTLVTVSSHuman germline sequences, light chain Human Germline 76EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPR VK3D-11, Jk2LLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQ RSNWHCTFGQGTKLEIKHuman Germline 77 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKVK1-39, Jk4 LLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ SYSFGGGTKVEIKHuman Germline 78 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKVK4-1, Jk2 PGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSFGQGTKLEIK Human Germline 79DIVLTQSPASLAVSPGQRATITCRASESVSFLGINLIHWYQQKPG VK7-3 (pseudogene),QPPKLLIYQASNKDTGVPARFSGSGSGTDFTLTINPVEANDTANY Jk1 YCLQSKNFPWTFGQGTKVEIK

In some embodiments, the antibody molecule comprises a VH T33V mutationrelative to A11. More specifically, in some embodiments, the anti-dengueantibody molecule comprises the CDR1 of the VH region of an antibody ofTable 1 (e.g., D88, F38, F108, or C88), using the Kabat or Chothiadefinitions of CDRs. In some embodiments, the anti-dengue antibodymolecule comprises the CDR1 and one or both of CDR2 and CDR3 of the VHregion of an antibody of Table 1 (e.g., D88, A48, F38, F108, or C88),using the Kabat or Chothia definitions of CDRs. In some embodiments, theanti-dengue antibody molecule comprises CDR1 of the VH region of anantibody of Table 1 (e.g., D88, A48, F38, F108, or C88) in combinationwith another 1, 2, 3, 4, or 5 (e.g., collectively 6) CDRs found in a VHand/or VL region of Table 2, using the Kabat of Chothia definitions ofCDRs. In some embodiments, the anti-dengue antibody molecule comprisesthe VH CDR1 of SEQ ID NO: 3. For instance, the anti-dengue antibodymolecule may comprise the VH CDR1 of SEQ ID NO: 3 in combination with aVH CDR2 and/or VHCDR3 of Table 3, e.g., VH CDR2 of SEQ ID NO: 4 and VHCDR3 of SEQ ID NO: 5. As a further example, the anti-dengue antibodymolecule may comprises the VH CDR1 of SEQ ID NO: 3 in combination withanother 1, 2, 3, 4, or 5 (e.g., collectively 6) CDRs found in a VHand/or VL region of Table 2.

In certain embodiments, the antibody molecule comprises a VH F65Lmutation relative to A11. In a Kabat-defined CDR of A11, position 65 isa CDR residue, while in a Chothia-defined CDR of A11, position 65 is aframework residue. In some embodiments, an antibody molecule's affinityfor dengue virus is unaffected by the F65L mutation. In someembodiments, the anti-dengue antibody molecule comprises the CDR2 of theVH region of an antibody of Table 1 (e.g., D88, A48, F38, F108, or C88),using the Kabat or Chothia definitions of CDRs. In some embodiments, theanti-dengue antibody molecule comprises the CDR2 and one or both of CDR1and CDR3 of the VH region of an antibody of Table 1 (e.g., D88, A48,F38, F108, or C88), using the Kabat or Chothia definitions of CDRs. Insome embodiments, the anti-dengue antibody molecule comprises CDR2 ofthe VH region of an antibody of Table 1 (e.g., D88, A48, F38, F108, orC88) in combination with another 1, 2, 3, 4, or 5 (e.g., collectively 6)CDRs found in a VH and/or VL region of Table 2, using the Kabat ofChothia definitions of CDRs. In some embodiments, the anti-dengueantibody molecule comprises the VH CDR2 of SEQ ID NO: 4. For instance,the anti-dengue antibody molecule may comprise the VH CDR2 of SEQ ID NO:4 in combination with a VH CDR1 and/or VH CDR3 of Table 3, e.g., VH CDR1of SEQ ID NO: 3 and VH CDR3 of SEQ ID NO: 5. As a further example, theanti-dengue antibody molecule may comprises the VH CDR2 of SEQ ID NO: 4in combination with another 1, 2, 3, 4, or 5 (e.g., collectively 6) CDRsfound in a VH and/or VL region of Table 2. In certain embodiments, theantibody molecule comprises a VH F65L mutation and a VH T33V mutationrelative to A11.

In some embodiments, the anti-dengue antibody molecule comprises adeletion of the S (del26) at position 26 in the VH relative to A11. Insome embodiments, the antibody molecule comprises del26 mutation incombination with a VH T33V mutation and/or a VH F65L mutation. Incertain embodiments, the antibody molecule comprises a del26 mutationand one or more CDRs of Table 3. In certain embodiments, the antibodymolecule comprises a del26 mutation in combination with 1, 2, 3, 4, 5,or 6 CDRs, in a VH and/or VL region of Table 2, using the Kabat ofChothia definitions of CDRs.

As shown in Example 4 of International Application Publication No.WO2015/122995, the N-terminus of the heavy chain is tolerant tomutations. Accordingly, in some embodiments, positions 1-6 of the heavychain sequence have 1, 2, 3, 4, 5, or 6 mutations relative to anantibody of Table 1. In some embodiments, an antibody molecule has asubstitution, insertion, or deletion at one or more (e.g., all) ofresidues 2, 3, 5, or 6 of a heavy chain sequence in Table 2. In certainembodiments, the antibody molecule comprises a portion of a heavy chainsequence of Table 2, e.g., amino acid positions 2-117, 3-117, 4-117,5-117, 6-117, 8-117, or 10-117.

As shown in Example 5 of International Application Publication No.WO2015/122995, positions 27 and 28 in the VH are tolerant of mutations,and in some embodiments, a mutation to position 27 and/or 28 enhancesbinding. Accordingly, in some embodiments, one or both of positions 27and 28 have a mutation relative to an antibody of Table 1.

Example 5 of International Application Publication No. WO2015/122995also shows that position 98 in the VH is tolerant of mutations, and insome embodiments, a mutation to position 98 enhances binding.Accordingly, in some embodiments, position 98 has a mutation relative toan antibody of Table 1.

In some embodiments, the anti-dengue antibody molecule comprises a heavychain constant region, a light chain constant region, and heavy andlight chain variable regions of Table 2. In certain embodiments, theanti-dengue antibody molecule comprises a heavy chain constant region, alight chain constant region, and variable regions that comprise 1, 2, 3,4, 5, or 6 CDRs of Table 3.

In some embodiments, the heavy chain variable region is a heavy chainvariable region of Table 1, wherein residue 98 in the VH can be anyamino acid. In certain embodiments, residue 98 can be any unchargedamino acid. In some embodiments, position 98 can be A, V, or S. Example5 of International Application Publication No. WO2015/122995 shows thatantibodies having residue A, V, or S at position 98 have good binding toEDIII.

During the humanization process, various framework regions (e.g., VHFW1) can be back-mutated to contain residues from mouse antibodies A11or B11. More broadly, in some embodiments, the anti-dengue antibodymolecule comprises the sequence of all or a portion of a VH region ofTable 1. For instance, in some embodiments, the anti-dengue antibodymolecule comprises amino acids 5-117, 10-117, 15-117, 20-117, 25-117,30-117, or 32-117 of a VH region of Table 1. In some embodiments, theanti-dengue antibody molecule comprises a VH FW1 region selected from amouse VH FW1 region (e.g., that found in A11 or B11) or a human VH FW1region (e.g., one found in an antibody of Table 1 or a human germline VHFW1 sequence). In some embodiments, the VH FW1 region has no more than1, 2, 3, or 4 positions of non-identity relative to amino acids 1-31 ofa VH sequence of Table 1.

In some embodiments, the anti-dengue antibody molecule comprises a VHFW2 region of an antibody of Table 1. In some embodiments, the VH FW2region has no more than 1, 2, 3, or 4 positions of non-identity relativeto amino acids 37-50 of a VH sequence of Table 1. An antibody moleculecapable of cross-reacting with EDIII from more than one serotype ofdengue virus has several advantageous properties. For example, onetherapy can be used to treat or diagnose multiple serotypes of dengue.In addition, a physician need not determine which serotype infected apatient in order to determine the appropriate therapy. Accordingly, insome embodiments, the anti-dengue antibody molecule is capable ofindependently binding to two, three, four, or more dengue virusserotypes with high affinity. For instance, the antibody molecule mayindependently bind with high affinity to EDIII of DV-1 and DV-2; of DV-1and DV-3; of DV-1 and DV-4; of DV-2 and DV-3; of DV-2 and DV-4; of DV-3and DV-4; or DV-1 and DV-2 and DV-3; of DV-1 and DV-2 and DV-4; of DV-1and DV-3 and DV-4; of DV-2 and DV-3 and DV-4; or of DV-1 and DV-2 andDV-3 and DV-4. In certain embodiments, the antibody molecule canindependently bind with high affinity to EDIII of DV-4 and EDIII of oneor more other DV serotypes.

Each serotype of dengue virus mentioned above is a class containingnumerous strains. The antibody molecules described herein show a goodbreadth of reactivity, binding to multiple strains within differentserotypes. Accordingly, in some embodiments, an antibody molecule asdescribed herein binds to and/or neutralizes one or more (e.g., at least2, 3, 4, 5, 10, 15, or 20, 25, or 30 or more) dengue virus strains,e.g., strains selected from: DENV-4 BC2, DENV-4-Sing10, DENV-4 NewCal09,DENV-4 Phil56, DENV-4 Thailand/1997, DENV-3 Sing09, DENV-3 Nic10, DENV-4Brazil/2011, DENV-4 Venezuela/2008, DENV-4 Colombia/1997, DENV-3 H87,DENV-3 Puerto Rico/1977, DENV-3 Cambodia/2008, DENV-2 Peru95, DENV-2Sing08, DENV-2 NGC, DENV-2 Venezuela/2007, DENV-2 Vietnam/2007, DENV-1Hawaii/1944, DENV-2 New Guinea/1944 (NGC), DENV-3 Philippines/1956(H87), DENV-4 Mexico/1997 (BC287/97), DENV-4 H241 DENV-1 Thailand/1964,DENV-1 Mexico/2007, DENV-1 Vietnam/2008, and DENV-1 Malaysia/2005, thestrains listed in Table 6 herein (e.g., those strains for which EDIIIsequences are provided in Table 6), the strains deposited in the ATCC,the strains listed in the World Reference Center for Emerging Virusesand Arboviruses (WRCEVA) (available atwww.niaid.nih.gov/labsandresources/resources/dmid/wrceva/Pages/default.aspx),and the strains listed in the CDC's Division or Vector Borne InfectiousDiseases (available at www2a.cdc.gov/nczved/dvbid/misc/reg.asp).

In some embodiments, the antibody molecule binds with high affinity toone or more of DV-1, DV-2, DV-3, and DV-4. An EDIII amino acid sequenceof the E protein of each of these serotypes is, in some embodiments, anE protein sequence provided in Table 6.

In some embodiments, an antibody molecule disclosed herein does notactivate antibody-dependent enhancement (ADE). ADE is described in moredetail in Balsitis et al., Lethal Antibody Enhancement of Dengue Diseasein Mice Is Prevented by Fc Modification, PLoS Pathog 6(2): e1000790.doi:10.1371/journal.ppat.1000790. Briefly, ADE describes a situation inwhich a person experiences two sequential dengue infections with dengueviruses of different serotypes, and the occurrence of the firstinfection makes the second infection more severe (e.g., more likely toprogress into dengue hemorrhagic fever). A mechanism for ADE may be thatan anti-dengue antibody binds simultaneously to the virus and to anantibody Fc receptor on a host cell, increasing infectivity. As is clearfrom the FRNT experiments disclosed herein, this application providesnumerous antibody molecules that reduce, rather than increase,infectivity. Accordingly, in certain embodiments, an antibody moleculeas described herein does not activate ADE in a patient. In someembodiments, the antibody inhibits ADE that is induced by otherantibodies (e.g., the patient's endogenous antibodies).

In certain embodiments, the antibody molecule binds to a linear orconformational epitope on EDIII.

As used herein, the term “antibody molecule” refers to a proteincomprising at least one immunoglobulin variable domain sequence. Theterm antibody molecule includes, for example, full-length, matureantibodies and antigen-binding fragments of an antibody. For example, anantibody molecule can include a heavy (H) chain variable domain sequence(abbreviated herein as VH), and a light (L) chain variable domainsequence (abbreviated herein as VL). In another example, an antibodymolecule includes two heavy (H) chain variable domain sequences and twolight (L) chain variable domain sequence, thereby forming two antigenbinding sites, such as Fab, Fab′, F(ab′)2, Fc, Fd, Fd′, Fv, single chainantibodies (scFv for example), single variable domain antibodies,diabodies (Dab) (bivalent and bispecific), and chimeric (e.g.,humanized) antibodies, which may be produced by the modification ofwhole antibodies or those synthesized de novo using recombinant DNAtechnologies. These functional antibody fragments retain the ability toselectively bind with their respective antigen or receptor. Antibodiesand antibody fragments can be from any class of antibodies including,but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass(e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies. The antibodies can bemonoclonal or polyclonal. The antibody can also be a human, humanized,CDR-grafted, or in vitro generated antibody. The antibody can have aheavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3, orIgG4. The antibody can also have a light chain chosen from, e.g., kappaor lambda.

Examples of antigen-binding fragments include: (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a diabody(dAb) fragment, which consists of a VH domain; (vi) a camelid orcamelized variable domain; (vii) a single chain Fv (scFv), see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody.These antibody fragments may be obtained using any suitable method,including several conventional techniques known to those with skill inthe art, and the fragments can be screened for utility in the samemanner as are intact antibodies.

The term “antibody” includes intact molecules as well as functionalfragments thereof. Constant regions of the antibodies can be altered,e.g., mutated, to modify the properties of the antibody (e.g., toincrease or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,or complement function).

The antibodies disclosed herein can also be single domain antibodies.Single domain antibodies can include antibodies whose complementarydetermining regions are part of a single domain polypeptide. Examplesinclude, but are not limited to, heavy chain antibodies, antibodiesnaturally devoid of light chains, single domain antibodies derived fromconventional 4-chain antibodies, engineered antibodies and single domainscaffolds other than those derived from antibodies. Single domainantibodies may be any of the art, or any future single domainantibodies. Single domain antibodies may be derived from any speciesincluding, but not limited to mouse, human, camel, llama, fish, shark,goat, rabbit, and bovine. According to some aspects, a single domainantibody is a naturally occurring single domain antibody known as heavychain antibody devoid of light chains. Such single domain antibodies aredisclosed in WO 9404678, for example. For clarity reasons, this variabledomain derived from a heavy chain antibody naturally devoid of lightchain is known herein as a VHH or nanobody to distinguish it from theconventional VH of four chain immunoglobulins. Such a VHH molecule canbe derived from antibodies raised in Camelidae species, for example incamel, llama, dromedary, alpaca and guanaco. Other species besidesCamelidae may produce heavy chain antibodies naturally devoid of lightchain; such VHHs are also contemplated.

The VH and VL regions can be subdivided into regions ofhypervariability, termed “complementarity determining regions” (CDR),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDRs has beenprecisely defined by a number of methods (see, Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and theAbM definition used by Oxford Molecular's AbM antibody modelingsoftware. See, generally, e.g., Protein Sequence and Structure Analysisof Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.:Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). In someembodiments, the following definitions are used: AbM definition of CDR1of the heavy chain variable domain and Kabat definitions for the otherCDRs. In certain embodiments, Kabat definitions are used for all CDRs.In addition, embodiments described with respect to Kabat or AbM CDRs mayalso be implemented using Chothia hypervariable loops. Each VH and VLtypically includes three CDRs and four FRs, arranged from amino-terminusto carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may or may not include one, two, or more N- orC-terminal amino acids, or may include other alterations that arecompatible with formation of the protein structure.

The term “antigen-binding region” refers to the part of an antibodymolecule that comprises determinants that form an interface that bindsto an E protein, or an epitope thereof. With respect to proteins (orprotein mimetics), the antigen-binding region typically includes one ormore loops (of at least, e.g., four amino acids or amino acid mimics)that form an interface that binds to the E protein. Typically, theantigen-binding region of an antibody molecule includes at least one ortwo CDRs, or more typically at least three, four, five or six CDRs.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope. Amonoclonal antibody can be made by hybridoma technology or by methodsthat do not use hybridoma technology (e.g., recombinant methods).

An “effectively human” protein is a protein that does not evoke aneutralizing antibody response, e.g., the human anti-murine antibody(HAMA) response. HAMA can be problematic in a number of circumstances,e.g., if the antibody molecule is administered repeatedly, e.g., intreatment of a chronic or recurrent disease condition. A HAMA responsecan make repeated antibody administration potentially ineffectivebecause of an increased antibody clearance from the serum (see, e.g.,Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and alsobecause of potential allergic reactions (see, e.g., LoBuglio et al.,Hybridoma, 5:5117-5123 (1986)).

The antibody molecule can be a polyclonal or a monoclonal antibody. Insome embodiments, the antibody can be recombinantly produced, e.g.,produced by any suitable phage display or combinatorial methods.

Various phage display and combinatorial methods for generatingantibodies are known in the art (as described in, e.g., Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO92/18619; Dower et al. International Publication No. WO 91/17271; Winteret al. International Publication WO 92/20791; Markland et al.International Publication No. WO 92/15679; Breitling et al.International Publication WO 93/01288; McCafferty et al. InternationalPublication No. WO 92/01047; Garrard et al. International PublicationNo. WO 92/09690; Ladner et al. International Publication No. WO90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contentsof all of which are incorporated by reference herein).

In some embodiments, the antibody is a fully human antibody (e.g., anantibody made in a mouse which has been genetically engineered toproduce an antibody from a human immunoglobulin sequence), or anon-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g.,monkey), camel antibody. In certain embodiments, the non-human antibodyis a rodent (mouse or rat antibody). Methods of producing rodentantibodies are known in the art.

Human monoclonal antibodies can be generated using transgenic micecarrying the human immunoglobulin genes rather than the mouse system.Splenocytes from these transgenic mice immunized with the antigen ofinterest are used to produce hybridomas that secrete human mAbs withspecific affinities for epitopes from a human protein (see, e.g., Woodet al. International Application WO 91/00906, Kucherlapati et al. PCTpublication WO 91/10741; Lonberg et al. International Application WO92/03918; Kay et al. International Application 92/03917; Lonberg, N. etal. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet.7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon etal. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol21:1323-1326).

An antibody can be one in which the variable region, or a portionthereof, e.g., the CDRs, are generated in a non-human organism, e.g., arat or mouse. Chimeric, CDR-grafted, and humanized antibodies are alsocontemplated. Antibodies generated in a non-human organism, e.g., a rator mouse, and then modified, e.g., in the variable framework or constantregion, to decrease antigenicity in a human are also contemplated.

Chimeric antibodies can be produced by any suitable recombinant DNAtechnique. Several are known in the art (see Robinson et al.,International Patent Publication PCT/US86/02269; Akira, et al., EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al., European Patent Application 173,494; Neubergeret al., International Application WO 86/01533; Cabilly et al. U.S. Pat.No. 4,816,567; Cabilly et al., European Patent Application 125,023;Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.(1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005;Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. NatlCancer Inst. 80:1553-1559).

A humanized or CDR-grafted antibody will have at least one or two butgenerally all three recipient CDRs (of heavy and or light immunoglobulinchains) replaced with a donor CDR. The antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDRs may bereplaced with non-human CDRs. It is only necessary to replace the numberof CDRs required for binding of the humanized antibody to EDIII. In someembodiments, the donor will be a rodent antibody, e.g., a rat or mouseantibody, and the recipient will be a human framework or a humanconsensus framework. Typically, the immunoglobulin providing the CDRs iscalled the “donor” and the immunoglobulin providing the framework iscalled the “acceptor.” In some embodiments, the donor immunoglobulin isa non-human (e.g., rodent). The acceptor framework is typically anaturally-occurring (e.g., a human) framework or a consensus framework,or a sequence about 85% or higher, e.g., 90%, 95%, 99% or higheridentical thereto.

As used herein, the term “consensus sequence” refers to the sequenceformed from the most frequently occurring amino acids (or nucleotides)in a family of related sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofproteins, each position in the consensus sequence is occupied by theamino acid occurring most frequently at that position in the family. Iftwo amino acids occur equally frequently, either can be included in theconsensus sequence. A “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence.

An antibody can be humanized by any suitable method, and several suchmethods known in the art (see e.g., Morrison, S. L., 1985, Science229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen etal. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No.5,693,762, the contents of all of which are hereby incorporated byreference).

Humanized or CDR-grafted antibodies can be produced by CDR-grafting orCDR substitution, wherein one, two, or all CDRs of an immunoglobulinchain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al.1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidleret al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539,the contents of all of which are hereby expressly incorporated byreference. Winter describes a CDR-grafting method which may be used toprepare humanized antibodies (UK Patent Application GB 2188638A, filedon Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of whichis expressly incorporated by reference.

Also provided are humanized antibodies in which specific amino acidshave been substituted, deleted or added. Criteria for selecting aminoacids from the donor are described in, e.g., U.S. Pat. No. 5,585,089,e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of whichare hereby incorporated by reference. Other techniques for humanizingantibodies are described in Padlan et al. EP 519596 A1, published onDec. 23, 1992.

The antibody molecule can be a single chain antibody. A single-chainantibody (scFV) may be engineered (see, for example, Colcher, D. et al.(1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin CancerRes 2:245-52). The single chain antibody can be dimerized ormultimerized to generate multivalent antibodies having specificities fordifferent epitopes of the same target protein.

In some embodiments, the antibody molecule has a heavy chain constantregion chosen from, e.g., the heavy chain constant regions of IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosenfrom, e.g., the (e.g., human) heavy chain constant regions of IgG1,IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has alight chain constant region chosen from, e.g., the (e.g., human) lightchain constant regions of kappa or lambda. The constant region can bealtered, e.g., mutated, to modify the properties of the antibody (e.g.,to increase or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,and/or complement function). In some embodiments the antibody haseffector function and can fix complement. In other embodiments theantibody does not recruit effector cells or fix complement. In certainembodiments, the antibody has reduced or no ability to bind an Fcreceptor. For example, it may be an isotype or subtype, fragment orother mutant, which does not support binding to an Fc receptor, e.g., ithas a mutagenized or deleted Fc receptor binding region.

The antibody constant region is altered in some embodiments. Methods foraltering an antibody constant region are known in the art. Antibodieswith altered function, e.g. altered affinity for an effector ligand,such as FcR on a cell, or the C1 component of complement can be producedby replacing at least one amino acid residue in the constant portion ofthe antibody with a different residue (see e.g., EP 388,151 A1, U.S.Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all ofwhich are hereby incorporated by reference) Amino acid mutations whichstabilize antibody structure, such as S228P (EU nomenclature, S241P inKabat nomenclature) in human IgG4 are also contemplated. Similar type ofalterations could be described which if applied to the murine, or otherspecies immunoglobulin would reduce or eliminate these functions.

In some embodiments, the only amino acids in the anti-dengue antibodymolecule are canonical amino acids. In some embodiments, the anti-dengueantibody molecule comprises naturally-occurring amino acids; analogs,derivatives and congeners thereof; amino acid analogs having variantside chains; and/or all stereoisomers of any of any of the foregoing.The anti-dengue antibody molecule may comprise the D- or L-opticalisomers of amino acids and peptidomimetics.

A polypeptide of an anti-dengue antibody molecule may be linear orbranched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The antibody molecule may also bemodified; for example, by disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation,such as conjugation with a labeling component. The polypeptide can beisolated from natural sources, can be a produced by recombinanttechniques from a eukaryotic or prokaryotic host, or can be a product ofsynthetic procedures.

The anti-dengue antibody molecule can be used alone in unconjugatedform, or can be bound to a substance, e.g., a toxin or moiety (e.g., atherapeutic drug; a compound emitting radiation; molecules of plant,fungal, or bacterial origin; or a biological protein (e.g., a proteintoxin) or particle (e.g., a recombinant viral particle, e.g., via aviral coat protein). For example, the anti-dengue antibody can becoupled to a radioactive isotope such as an α-, β-, or γ-emitter, or aβ- and γ-emitter.

An antibody molecule can be derivatized or linked to another functionalmolecule (e.g., another peptide or protein). As used herein, a“derivatized” antibody molecule is one that has been modified. Methodsof derivatization include but are not limited to the addition of afluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinityligand such as biotin. Accordingly, the antibody molecules are intendedto include derivatized and otherwise modified forms of the antibodiesdescribed herein, including immunoadhesion molecules. For example, anantibody molecule can be functionally linked (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a toxin, a pharmaceuticalagent, and/or a protein or peptide that can mediate association of theantibody or antibody portion with another molecule (such as astreptavidin core region or a polyhistidine tag).

Some types of derivatized antibody molecule are produced by crosslinkingtwo or more antibodies (of the same type or of different types, e.g., tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an anti-dengue antibody molecule maybe derivatized (or labeled) to include fluorescent compounds, variousenzymes, prosthetic groups, luminescent materials, bioluminescentmaterials, fluorescent emitting metal atoms, e.g., europium (Eu), andother anthanides, and radioactive materials (described below). Exemplaryfluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5dimethylamine-1-napthalenesulfonyl chloride,phycoerythrin and the like. An antibody may also be derivatized withdetectable enzymes, such as alkaline phosphatase, horseradishperoxidase, β-galactosidase, acetylcholinesterase, glucose oxidase andthe like. When an antibody is derivatized with a detectable enzyme, itis detected by adding additional reagents that the enzyme uses toproduce a detectable reaction product. For example, when the detectableagent horseradish peroxidase is present, the addition of hydrogenperoxide and diaminobenzidine leads to a colored reaction product, whichis detectable. An antibody molecule may also be derivatized with aprosthetic group (e.g., streptavidin/biotin and avidin/biotin). Forexample, an antibody may be derivatized with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Examplesof suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; an example of aluminescent material includes luminol; and examples of bioluminescentmaterials include luciferase, luciferin, and aequorin.

Labeled antibody molecule can be used, for example, diagnosticallyand/or experimentally in a number of contexts, including (i) to isolatea predetermined antigen by standard techniques, such as affinitychromatography or immunoprecipitation; (ii) to detect a predeterminedantigen (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the protein; (iii)to monitor protein levels in tissue as part of a clinical testingprocedure, e.g., to determine the efficacy of a given treatment regimen.

An antibody molecule may be conjugated to another molecular entity,typically a label or a therapeutic (e.g., immunomodulatory,immunostimularoty, cytotoxic, or cytostatic) agent or moiety.Radioactive isotopes can be used in diagnostic or therapeuticapplications. Radioactive isotopes that can be coupled to theanti-dengue antibodies include, but are not limited to α-, β-, orγ-emitters, or β- and γ-emitters. Such radioactive isotopes include, butare not limited to iodine (¹³¹I or ¹²⁵I), yttrium (⁹⁰Y), lutetium(¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium, astatine (²¹¹At), rhenium(¹⁸⁶Re) bismuth (²¹²Bi or ²¹³Bi), indium (¹¹¹In), technetium (⁹⁹ mTc),phosphorus (³²P), rhodium (¹⁸⁸Rh), sulfur (³⁵S), carbon (¹⁴C), tritium(³H), chromium (⁵¹Cr), chlorine (³⁶Cl), cobalt (⁵⁷Co or ⁵⁸Co), iron(⁵⁹Fe), selenium (⁷⁵Se), or gallium (⁶⁷Ga). Radioisotopes useful astherapeutic agents include yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium(²²⁵Ac), praseodymium, astatine (²¹¹At), rhenium (¹⁸⁶Re), bismuth (²¹²Bior ²¹³Bi) and rhodium (¹⁸⁸Rh). Radioisotopes useful as labels, e.g., foruse in diagnostics, include iodine (¹³¹I or ¹²⁵I), indium (¹¹¹In)technetium (⁹⁹mTc), phosphorus (³²P), carbon (¹⁴C), and tritium (³H), orone or more of the therapeutic isotopes listed above.

The present disclosure provides radiolabeled antibody molecules andmethods of labeling the same. In some embodiments, a method of labelingan antibody molecule is disclosed. The method includes contacting anantibody molecule, with a chelating agent, to thereby produce aconjugated antibody. The conjugated antibody is radiolabeled with aradioisotope, e.g., ¹¹¹Indium, ⁹⁰Yttrium and ¹⁷⁷Lutetium, to therebyproduce a labeled antibody molecule.

As is discussed above, the antibody molecule can be conjugated to atherapeutic agent. Therapeutically active radioisotopes have alreadybeen mentioned. Examples of other therapeutic agents include anti-viralagents.

In some aspects, this disclosure provides a method of providing anantibody molecule disclosed herein. The method includes: providing anantigen, e.g., a dengue virus E protein or portion thereof; obtaining anantibody molecule that specifically binds to the antigen; evaluatingefficacy of the antibody molecule in modulating activity of the antigenand/or organism expressing the antigen, e.g., dengue virus. The methodcan further include administering the antibody molecule, including aderivative thereof (e.g., a humanized antibody molecule) to a subject,e.g., a human.

This disclosure provides an isolated nucleic acid molecule encoding theabove antibody molecule, vectors and host cells thereof. The nucleicacid molecule includes but is not limited to RNA, genomic DNA and cDNA.

Formulations

A formulation of an antibody molecule can include, e.g., an anti-dengueantibody molecule described herein and a buffer. The pH of theformulation is generally pH 5.5-7.0.

In some embodiments, the formulation is a liquid formulation. In someembodiments, the formulation is stored as a liquid. In otherembodiments, the formulation is a lyophilized formulation. In certainembodiments, the formulation is prepared as a liquid and then is dried,e.g., by lyophilization or spray-drying, prior to storage. A driedformulation can be used as a dry compound, e.g., as an aerosol orpowder, or reconstituted to its original or another concentration, e.g.,using water, a buffer, or other appropriate liquid.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized protein formulation in a diluent such that theprotein is dispersed in the reconstituted formulation. The reconstitutedformulation in suitable for administration (e.g. parenteraladministration) to a patient to be treated with the protein of interestand, in certain embodiments of the invention, may be one which issuitable for subcutaneous administration. The “diluent” of interestherein is one which is pharmaceutically acceptable (safe and non-toxicfor administration to a human) and is useful for the preparation of areconstituted formulation. Exemplary diluents include sterile water,bacteriostatic water for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest, significantly prevents or reduces chemical and/or physicalinstability of the protein upon lyophilization and subsequent storage.Exemplary lyoprotectants that can be used in the formulations describedherein include, but are not limited to, sugars such as sucrose ortrehalose; an amino acid such as monosodium glutamate or histidine; amethylamine such as betaine; a lyotropic salt such as magnesium sulfate;a polyol such as trihydric or higher sugar alcohols, e.g. glycerin,erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol;propylene glycol; polyethylene glycol; pluronics; and combinationsthereof. Typically, the lyoprotectant is a non-reducing sugar, such astrehalose or sucrose. The lyoprotectant is added to the pre-lyophilizedformulation in a “lyoprotecting amount” which means that, followinglyophilization of the protein in the presence of the lyoprotectingamount of the lyoprotectant, the protein essentially retains itsphysical and chemical stability and integrity upon lyophilization andstorage.

A “bulking agent” is a compound which adds mass to the lyophilizedmixture and contributes to the physical structure of the lyophilizedcake (e.g. facilitates the production of an essentially uniformlyophilized cake which maintains an open pore structure). Exemplarybulking agents that can be used in the formulations described hereininclude, but are not limited to, mannitol, glycine, polyethylene glycolor xorbitol.

In some embodiments, the anti-dengue antibody molecule purificationprocess is designed to permit transfer of an anti-dengue antibodymolecule into a formulation suitable for storage as a liquid. In otherembodiments, the anti-dengue antibody molecule purification process isdesigned to permit transfer of an anti-dengue antibody molecule into aformulation for long-term storage as a frozen liquid and subsequentlyfor freeze-drying.

In some embodiments, the formulation is lyophilized with the protein ata specific concentration. The lyophilized formulation can then bereconstituted as needed with a suitable diluent (e.g., water) toresolubilize the original formulation components to a desiredconcentration, generally the same or higher concentration compared tothe concentration prior to lyophilization. The lyophilized formulationmay be reconstituted to produce a formulation that has a concentrationthat differs from the original concentration (i.e., beforelyophilization), depending upon the amount of water or diluent added tothe lyophilate relative to the volume of liquid that was originallyfreeze-dried. Suitable formulations can be identified by assaying one ormore parameters of antibody integrity. The assayed parameters aregenerally the percentage of HMW species or the percentage of LMWspecies.

The percentage of HMW species or LMW species is determined either as apercentage of the total protein content in a formulation, asconcentration in the total formulation, or as a change in the percentageincrease over time (i.e., during storage). The total percentage of HMWspecies in an acceptable formulation is not greater than 10% (e.g., notgreater than 5%, not greater than 4%, not greater than 3%, not greaterthan 2%, or not greater than 1%) HMW species after storage as alyophilate or liquid at 2° C. to 50° C. (e.g., at 4° C. to 45° C., at 5°C. to 40° C., at 5° C. to 25° C., at 25° C. to 40° C., at 4° C. to 25°C., at 4° C. to 15° C., at about 4° C., at about 5° C., at about 25° C.,at about 40° C., or at about 45° C.) for at least one week, two weeks,one month, three months, six months, nine months, or one year or notgreater than about 10% LMW species after storage as a lyophilate orliquid at 2° C. to 50° C. (e.g., at 4° C. to 45° C., at 5° C. to 40° C.,at 5° C. to 25° C., at 25° C. to 40° C., at 4° C. to 25° C., at 4° C. to15° C., at about 4° C., at about 5° C., at about 25° C., at about 40°C., or at about 45° C.) for at least one week, two weeks, one month,three months, six months, nine months, or one year. In an embodiment,the total percentage of HMW species is not greater than 5%. In anotherembodiment, the total percentage of HMW species is not greater than 3%.By “about” is meant ±20% of a cited numerical value. Thus, for example,“about 20° C.” means 16° C. to 24° C.

Typically, the stability profile is less than 10% HMW/LMW at 2°−8° C.for a refrigerated product, and 25° C. for a room-temperature product.HMW species or LMW species are assayed in a formulation stored as alyophilate after the lyophilate is reconstituted. 40° C. or 45° C. is anaccelerated condition that is generally used for testing stability anddetermining stability for short-term exposures to non-storageconditions, e.g., as may occur during transfer of a product duringshipping. In some embodiments 40° C. is used.

When the assayed parameter is the percentage change in HMW species orLMW species, the percent of total protein in one or both species afterstorage is compared to the percent total protein in one or both speciesprior to storage (e.g., upon preparation of the formulation). Thedifference in the percentages can be determined. In general, the changein the percentage of protein in HMW species or LMW species in liquidformulations is not greater than 10%, e.g., not greater than about 8%,not greater than about 7%, not greater than about 6%, not greater thanabout 5%, not greater than about 4%, or not greater than about 3% afterstorage at 2° C.-8° C. (e.g., 4° C.) or 25° C., for about one week, twoweeks, one month, three months, six months, nine months, or twelvemonths, eighteen, or twenty-four months. By “about” is meant ±20% of acited numerical value. Thus, about 10% means 8% to 12%. Formulationsstored as lyophilized product generally have less than about 5%, lessthan about 4%, less than about 3%, or less than about 2% HMW species orless than about 5%, less than about 4%, less than about 3%, or less thanabout 2% LMW species after reconstitution following storage at 2° C.-8°C. (e.g., 4° C.) or 25° C. for about for about one week, two weeks, onemonth, three months, six months, nine months, or twelve months,eighteen, or twenty-four months.

Formulations of anti-dengue antibody molecules can be stored as a liquidfor, e.g., at least two weeks, at least one month, at least two months,at least three months, at least four months, at least six months, atleast nine months, at least one year, or at least two years.Formulations of anti-dengue antibody molecules can be stored as alyophilate for, e.g., at least two years, at least three years, at leastfour years, or at least five years.

Additional details related to components of formulations and methods ofassaying the integrity of the anti-dengue antibody molecule, e.g., theanti-dengue antibody molecule described herein, in a formulation areprovided infra.

In some embodiments, the formulations described herein are formulatedfor parenteral or intravenous administration.

Anti-dengue antibody molecule concentrations in the formulationsdescribed herein are generally between about 5 mg/mL to about 250 mg/mL,e.g., about 5 mg/mL to about 200 mg/mL, about 5 mg/mL to about 150mg/mL, about 5 mg/mL to about 100 mg/mL, about 5 mg/mL to about 50mg/mL, about 10 mg/mL to about 40 mg/mL, about 20 mg/mL to about 30mg/mL, about 50 mg/mL to about 100 mg/mL, or about 150 mg/mL to about200 mg/mL, e.g., about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20mg/mL, about 25 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL,about 75 mg/mL, about 100 mg/mL, about 150 mg/mL, about 200 mg/mL, orabout 250 mg/mL.

In some embodiments, the antibody molecule is present at a concentrationof about 10 to about 40 mg/mL, e.g., about 20 to about 30 mg/mL, e.g.,about 25 mg/mL. In an embodiment, the antibody molecule is present at aconcentration of about 25 mg/mL.

In the context of ranges, “about” means −20% of the lower-citednumerical value of the range and +20% of the upper-cited numerical valueof the range. In the context of ranges, e.g., about 10 mg/mL to about100 mg/mL, this means, between 8 mg/mL to 120 mg/mL. In some cases,antibody concentrations in formulations can be, for example, between 1mg/mL and 100 mg/mL, e.g., 2 mg/mL and 80 mg/mL, 5 mg/mL and 60 mg/mL,10 mg/mL and 50 mg/mL, 15 mg/mL and 40 mg/mL, 20 mg/mL and 30 mg/mLanti-dengue antibody molecule described herein, e.g., Ab D88. Suchantibody formulations can be used as therapeutic agents. Accordingly,the concentration of anti-dengue antibody molecule in a formulation issufficient to provide such dosages in a volume of the formulation thatis tolerated by a subject being treated and is appropriate for themethod of administration. In one non-limiting example, to supply a highdosage subcutaneously, in which the volume limitation is small (e.g.,about 1 ml to 1.2 ml per injection), the concentration of antibody isgenerally at least 25 mg/mL or greater, e.g., 100 mg/mL or greater,e.g., 100 mg/mL to 500 mg/mL, 100 mg/mL to 250 mg/mL, or 100 mg/mL to150 mg/mL. Such high concentrations can be achieved, for example, byreconstituting a lyophilized formulation in an appropriate volume ofdiluent (e.g., sterile water for injection, buffered saline). In somecases, the reconstituted formulation has a concentration of between 25mg/mL and 500 mg/mL, e.g., between about 100 mg/mL and 500 mg/mL (e.g.,100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, 250 mg/mL, 275mg/mL, 300 mg/mL, 350 mg/mL, 375 mg/mL, 400 mg/mL, 425 mg/mL, 450 mg/mL,475 mg/mL and 500 mg/mL). For delivery via inhalation, the formulationis generally somewhat concentrated (e.g., between about 25 mg/mL and 500mg/mL, e.g., between about 100 mg/mL and 500 mg/mL) so as to provide asufficient dose in a limited volume of aerosol for inspiration. In somecases, low concentrations (e.g., between about 0.05 mg/mL and 1 mg/mL)are used. Methods are known in the art to adapt the dosage delivered tothe method of delivery, e.g., a jet nebulizer or a metered aerosol.

Buffers

The pH of a formulation as described herein is generally between aboutpH 5.0 to about 7.5, for example, about pH 5.5 to about 6.5, about pH5.5 to about 6.0, about pH 6.0 to about 6.5, about pH 6.5 to about 7.0,pH 5.5, pH 6.0, pH 6.5 or pH 7.0. In general, a buffer that can maintaina solution at pH 5.5 to pH 7.0 is used to prepare a formulation.Suitable buffers include, without limitation, citric acid, HEPES,histidine, arginine, potassium acetate, potassium citrate, potassiumphosphate (K₂HPO₄), sodium acetate, sodium bicarbonate, sodium citrate,sodium phosphate (NaH₂PO₄), Tris base, and Tris-HCl. The concentrationof the buffer is between about 5 mM and about 100 mM, e.g., about 5 mMto about 50 mM. In some cases, sodium phosphate buffer is used at aconcentration of about 25 mM. In some cases, sodium citrate buffer isused at a concentration of about 10 mM or 25 mM. In other cases,histidine buffer is used at a concentration of about 25 mM. In somecases, arginine buffer is used at a concentration of about 25 mM.

Tonicity Agents

Tonicity agents are known in the art and include, e.g., dextrose,glycerin, mannitol, potassium chloride, and sodium chloride.

The tonicity agent is generally used at a concentration of about 50 mMto about 200 mM. For example, the tonicity agent can be used at aconcentration of about 50 mM to about 200 mM, e.g., about 60 mM to about190 mM, about 70 mM to about 180 mM, about 80 mM to about 170 mM, about90 mM to about 160 mM, about 100 mM to about 150 mM, about 145 mM toabout 155 mM, about 140 mM to about 160 mM, about 135 mM to about 165mM, about 130 mM to about 170 mM, about 120 mM to about 180 mM, about 70mM to about 130 mM, about 80 to about 120 mM, about 90 mM to about 110mM, about 110 mM to about 190 mM, about 100 mM to about 200 mM, about 50mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM to about 80mM, about 100 mM to about 150 mM, or about 150 mM to about 120 mM, e.g.,about 200 mM or less, about 160 mM or less about 150 mM or less, about120 mM or less, about 110 mM or less, about 100 mM or less, about 80 orless, or about 75 mM or less, e.g., about 50 mM, about 60 mM, about 70mM, about 75 mM about 80 mM, about 90 mM, about 100 mM, about 110 mM,about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM,about 170 mM, about 180 mM, about 190 mM, or about 200 mM.

In an embodiment, the tonicity agent is used at a concentration of about50 mM to about 200 mM, about 75 mM to about 150 mM, about 120 mM toabout 180 mM, e.g., about 140 mM to about 160 mM, e.g., about 150 mM. Inanother embodiment, the tonicity agent is used at a concentration ofabout 50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM toabout 80 mM, e.g., about 75 mM. In another embodiment, the tonicityagent is used at a concentration of about 50 mM to about 200 mM, about60 mM to about 130 mM, about 70 mM to about 120 mM, about 80 mM to about110 mM, e.g., about 100 mM. In an embodiment, the tonicity agentcomprises sodium chloride. In an embodiment, the tonicity agentcomprises sodium chloride and is used at a concentration of about 140 mMto about 160 mM, e.g., about 150 mM. In an embodiment, the tonicityagent comprises sodium chloride and is used at a concentration of about55 mM to about 95 mM, e.g., about 75 mM. In an embodiment, the tonicityagent comprises sodium chloride and is used at a concentration of about80 mM to about 120 mM, e.g., about 100 mM.

In an embodiment, the tonicity agent provides a tonicity (or osmolality)of about 150 mOsm/kg to about 400 mOsm/kg, 180 mOsm/kg to about 400mOsm/kg, about 190 mOsm/kg to about 390 mOsm/kg, about 200 mOsm/kg toabout 380 mOsm/kg, about 210 mOsm/kg to about 370 mOsm/kg, about 220mOsm/kg to about 360 mOsm/kg, about 230 mOsm/kg to about 350 mOsm/kg,about 240 mOsm/kg to about 340 mOsm/kg, about 250 mOsm/kg to about 330mOsm/kg, about 260 mOsm/kg to about 320 mOsm/kg, about 270 mOsm/kg toabout 310 mOsm/kg, about 280 mOsm/kg to about 300 mOsm/kg, or about 285mOsm/kg to about 295 mOsm/kg, e.g., about 240 mOsm/kg, about 250mOsm/kg, about 260 mOsm/kg, about 270 mOsm/kg, about 280 mOsm/kg, about290 mOsm/kg, about 300 mOsm/kg, about 310 mOsm/kg, about 320 mOsm/kg,about 330 mOsm/kg, or about 340 mOsm/kg. In an embodiment, the tonicityagent provides a tonicity (osmolality) of about 150 mOsm/kg to about 400mOsm/kg, about 200 mOsm/kg to about 350 mOsm/kg, or about 250 mOsm/kg toabout 300 mOsm/kg.

The tonicity agent used in the formulation can generally provide atonicity (or osmolarity) of about 150 mOsm/L to about 400 mOsm/L, about160 mOsm/L to about 390 mOsm/L, about 170 mOsm/L to about 380 mOsm/L,about 180 mOsm/L to about 370 mOsm/L, about 190 mOsm/L to about 360mOsm/L, about 200 mOsm/L to about 370 mOsm/L, about 210 mOsm/L to about360 mOsm/L, about 220 mOsm/L to about 350 mOsm/L, about 200 mOsm/L toabout 350 mOsm/L, about 220 mOsm/L to about 340 mOsm/L, or about 220mOsm/L to about 340 mOsm/L, about 230 mOsm/L to about 330 mOsm/L, orabout 240 mOsm/L to about 320 mOsm/L, e.g., about 250 mOsm/L, about 260mOsm/L, about 270 mOsm/L, about 280 mOsm/L, about 290 mOsm/L, about 300mOsm/L, about 310 mOsm/L, about 320 mOsm/L, about 330 mOsm/L, about 340mOsm/L, or about 350 mOsm/L.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

Surfactants

In certain embodiments, a surfactant is included in the formulation.Examples of surfactants include, without limitation, nonionicsurfactants such as polysorbates (e.g., polysorbate-20, polysorbate-40,polysorbate-60, polysorbate-65, polysorbate-80, or polysorbate-85);poloxamers (e.g., poloxamer 188); Triton™; sodium dodecyl sulfate (SDS);sodium laurel sulfate; sodium octyl glycoside; lauryl-sulfobetaine,myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine,lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine,stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine,lauroamidopropyl-betaine, cocamidopropyl-betaine,linoleamidopropyl-betaine, myristamidopropyl-betaine,palmidopropyl-betaine, isostearamidopropyl-betaine (e.g.lauroamidopropyl), myristarnidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the Monaquat™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, polyoxethylene-stearates, polyoxyethylene alkyl ethers, e.g. polyoxyelhylenemonolauryl ether, polyoxyethylene-polyoxypropylene copolymer (Poloxamer,Piuronic), and copolymers of ethylene and propylene glycol (e.g.pluronics, PF68). For example, the surfactant can be present in theformulation (liquid or prior to lyophilization) in an amount from about0.001% to 0.5%, e.g., from about 0.005% to 0.05%, about 0.005% to about0.2%, and about 0.01% to 0.2%. In one embodiment the nonionic surfactantis poiysorbate 80. In one embodiment the polysorbate 80 concentration isabout 0.001 to 0.05% (w/v). In one embodiment, the polysorbate 80concentration is about 0.02% (w/v).

Cryoprotectants

Cryoprotectants are known in the art and include, e.g., sucrose,trehalose, sorbitol and glycerol. A cryoprotectant exhibiting lowtoxicity in biological systems is generally used. The cryoprotectant isincluded in the formulation at a concentration of about 50 mM to about150 mM.

In some embodiments, sucrose is used at a concentration of about 50 mMto about 150 mM, about 60 mM to about 140 mM, about 70 mM to about 130mM, about 80 mM to about 120 mM, about 90 mM to about 110 mM, about 50mM to about 90 mM, about 60 mM to about 80 mM, e.g., about 200 mM orless, about 190 mM or less, about 180 mM or less, about 170 mM or less,about 160 mM or less, about 150 mM or less, about 140 mM or less, about130 mM or less, about 120 mM or less, about 110 mM or less, about 100 mMor less, about 90 mM or less, about 80 mM or less, about 75 mM or less,or about 60 mM or less. In some embodiments, sucrose is used at aconcentration of about 75 mM. In some embodiments, sucrose is used at aconcentration of about 150 mM.

In some embodiments, sorbitol is used at a concentration of about 50 mMto about 150 mM, about 70 mM to about 140 mM, about 90 mM to about 130mM, about 150 mM or less, about 145 mM or less, about 140 mM or less,about 135 mM or less, about 130 mM or less, about 125 mM or less, about120 mM or less, about 115 mM or less, about 110 mM or less, about 105 mMor less, or about 100 mM or less. In some embodiments, sorbitol is usedat a concentration of about 125 mM.

Histidine buffer, which can be used as a buffer in an anti-dengueantibody molecule formulation, may have cryoprotectant properties. Insome embodiments of the invention, a histidine buffer is used inconjunction with a cryoprotectant such as a sugar, e.g., sucrose orsorbitol.

The viscosity of a formulation is generally one that is compatible withthe route of administration of the formulation. In some embodiments, theviscosity of the formulation is between 1 cP and 2 cP, or similar towater (about 1 cP). In other embodiments, the viscosity of theformulation is between about 5 cP and about 40 cP. In specificembodiments, the viscosity of the formulation is 1 cP, 2 cP, 3 cP, 4 cP,5 cP, 10 cP, 15 cP, 20 cP, 25 cP, 30 cP, 35 cP, or 40 cP.

Additions to Formulations

Formulations are stored as sterile solutions or sterile lyophilates.Prevention of the action of microorganisms in formulations can also beachieved by including at least one antibacterial and/or antifungal agentin a formulation, for example, parabens, chlorobutanol, phenol, ascorbicacid, thimerosal, and the like. In some cases, a lyophilate isreconstituted with bacteriostatic water (e.g., water containing 0.9%benzyl alcohol). Considerations for the inclusion of a preservative in aformulation are known in the art as are methods of identifyingpreservatives that are compatible with a specific formulation and methodof delivery (e.g., see Gupta, et al. (2003), AAPS Pharm. Sci. 5:article8, p. 1-9). A “preservative” is a compound which can be added to thediluent to essentially reduce bacterial action in the reconstitutedformulation, thus facilitating the production of a multi-usereconstituted formulation, for example. Examples of potentialpreservatives include octadecyldimethylbenzyl ammonium chloride,hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols such as phenol, butyl and benzylalcohol, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

In some cases, the formulation is isotonic. In general, any componentknown in the art that contributes to the solution osmolarity/tonicitycan be added to a formulation (e.g., salts, sugars, polyalcohols, or acombination thereof). Isotonicity is generally achieved using either acomponent of a basic formulation (such as sucrose) in an isotonicconcentration or by adding an additional component such as, a sugar, apolyalcohol such as manitol or sorbitol, or a salt such as sodiumchloride.

In some cases, a salt is used in an anti-dengue antibody moleculeformulation, e.g., to achieve isotonicity or to increase the integrityof the anti-dengue antibody molecule of the formulation. Salts suitablefor use are discussed, supra.

In certain embodiments the pharmaceutical formulation according to thepresent invention comprises a stabilizer (also known as a stabilizingagent). Stabilizers, include, but are not limited to human serum albumin(HSA), bovine serum albumin (BSA), casein, globulin, α-lactalbumin,lactate dehydrogenase (LDH), lysozyme, myoglobin, ovalbumin, and RNaseA. Exemplary stabilizers also include amino acids and their metabolites,such as, glycine, alanine (α-alanine, β-alanine), arginine, betaine,leucine, lysine, glutamic acid, aspartic acid, proline,4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines(alanopine, octopine, or strombine), and trimethylamine N-oxide (TMAO).In one embodiment, the stabilizer is an amino acid. In some embodiments,arginine is used. Exemplary stabilizers also include carbohydrates,e.g., sucrose, trehalose, mannitol, dextran, sorbitol, inositol,glucose, fructose, lactose, xylose, mannose, maltose, or raffinose.

Arginine is generally used at a concentration of about 50 mM to about200 mM. For example, arginine can be used at a concentration of about 50mM to about 200 mM, e.g., about 60 mM to about 190 mM, about 70 mM toabout 180 mM, about 80 mM to about 170 mM, about 90 mM to about 160 mM,about 100 mM to about 150 mM, about 145 mM to about 155 mM, about 140 mMto about 160 mM, about 135 mM to about 165 mM, about 130 mM to about 170mM, about 120 mM to about 180 mM, about 70 mM to about 130 mM, about 110mM to about 190 mM, about 100 mM to about 200 mM, about 50 mM to about100 mM, about 60 mM to about 90 mM, about 70 mM to about 80 mM, or about150 mM to about 120 mM, e.g., about 200 mM or less, about 160 mM or lessabout 150 mM or less, about 120 mM or less, about 110 mM or less, about100 mM or less, about 80 or less, or about 75 mM or less, e.g., about 50mM, about 60 mM, about 70 mM, about 75 mM about 80 mM, about 90 mM,about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM,about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, orabout 200 mM.

In an embodiment, arginine is used at a concentration of about 50 mM toabout 200 mM, about 75 mM to about 150 mM, about 120 mM to about 180 mM,e.g., about 140 mM to about 160 mM, e.g., about 150 mM. In anotherembodiment, arginine is used at a concentration of about 50 mM to about100 mM, about 60 mM to about 90 mM, about 70 mM to about 80 mM, e.g.,about 75 mM.

In certain embodiments the pharmaceutical formulation according to thepresent invention comprises a metal chelator. Metal chelators, include,but are not limited to EDTA and EGTA.

Methods of Making Formulations

In an aspect, the disclosure provides a method of making a formulationdescribed herein. The method includes combining an anti-dengue antibodymolecule described herein (e.g., D88) with a buffering agent describedherein, and one or both of a tonicity agent described herein or astabilizing agent described herein, optionally further with asurfactant, a carbohydrate, or both, as described herein, thereby makingthe formulation.

In an embodiment, the method comprises an anti-dengue antibody moleculedescribed herein with (a) a buffering agent (e.g., histidine or sodiumcitrate), (b) a tonicity agent (e.g., sodium chloride), and (c) astabilizing agent (e.g., arginine or sucrose), wherein the formulationcomprises 10 mg/mL to 50 mg/mL of the anti-dengue antibody molecule, 10mM to 50 mM of the buffering agent (e.g., histidine or sodium citrate),50 mM to 150 mM of the tonicity agent (e.g., sodium chloride), and 50 mMto 150 mM of the stabilizing agent (e.g., arginine or sucrose), andwherein the formulation has a pH of 6 to 7 (e.g., pH 6.5).

In an embodiment, the method further comprises combining the antibodymolecule with a surfactant, e.g., polysorbate 80. In an embodiment, theformulation comprises 0.01% to 0.05% (e.g., 0.02%) polysorbate 80.

Exemplary Formulations

Exemplary anti-dengue antibody molecule formulations are described inTable 7.

In an embodiment, the formulation comprises 10 mg/mL to 50 mg/mL of ananti-dengue antibody molecule described herein, 10 mM to 50 mM histidineor sodium citrate, 50 mM to 150 mM sodium chloride, and 50 mM to 150 mMarginine or sucrose, wherein the formulation has a pH of 6 to 7.

For example, formulations F1-F21 contains 25 mg/mL antibody D88;formulations F8-F17 contains 25 mM histidine pH 6.5; formulation F1contains 10 mM sodium citrate pH 6.0, formulations F2-F5 contains 25 mMsodium citrate pH 6.5; formulations F2, F5, F6, F7, and F12-F21 contain75 mM NaCl, formulation F1 contains 100 mM NaCl, formulations F8-F9contain 150 mM NaCl; formulations F18-F21 contain 25 mM arginine,formulations F2, F6, F12, and F13 contain 75 mM arginine, formulationsF3, F4, F10, and F11 contain 150 mM arginine; formulations F5 and F7contain 75 sucrose, formulations F14, F15, F18, and F19 contain 100 mMsucrose; formulation F1 has a pH of 6.0, formulations F2-F5 and F8-F17have a pH of 6.5, formulations F6-F7 and F18-F21 have a pH of 7.0.

In an embodiment, the antibody molecule comprises:

(a) a heavy chain (HC) immunoglobulin variable region segmentcomprising:

an HC CDR1 comprising the sequence DVYMS (SEQ ID NO: 3),

an HC CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4), and

an HC CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5); and

(b) a light chain (LC) immunoglobulin variable region segmentcomprising:

an LC CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6),

an LC CDR2 comprising the sequence RASELQW (SEQ ID NO: 7), and

an LC CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8).

In an embodiment, the formulation comprises the heavy chain variableregion (VH) amino acid sequence of SEQ ID NO: 1. In an embodiment, theformulation comprises the light chain variable region (VL) amino acidsequence of SEQ ID NO: 2. In an embodiment, the formulation comprisesthe VH amino acid sequence of SEQ ID NO: 1 and the VL amino acidsequence of SEQ ID NO: 2.

In an embodiment, the formulation comprises 10 mg/mL to 40 mg/mL of theantibody molecule. In an embodiment, the formulation comprises 25 mg/mLof the antibody molecule.

In an embodiment, the formulation comprises 10 mM to 50 mM histidine. Inan embodiment, the formulation comprises 20 mM to 40 mM histidine. In anembodiment, the formulation comprises 25 mM histidine.

In an embodiment, the formulation comprises 10 mM to 50 mM sodiumcitrate. In an embodiment, the formulation comprises 10 mM to 30 mMsodium citrate. In an embodiment, the formulation comprises 25 mM sodiumcitrate.

In an embodiment, the formulation comprises 75 mM to 150 mM sodiumchloride. In an embodiment, the formulation comprises 75 mM sodiumchloride.

In an embodiment, the formulation comprises 50 mM to 150 mM arginine. Inan embodiment, the formulation comprises 75 mM to 150 mM arginine. In anembodiment, the formulation comprises 75 mM arginine.

In an embodiment, the formulation comprises 50 mM to 150 mM sucrose. Inan embodiment, the formulation comprises 75 mM to 100 mM sucrose. In anembodiment, the formulation comprises 100 mM sucrose.

In an embodiment, the formulation has a pH of 6.5.

In an embodiment, the formulation further comprises polysorbate 80. Inan embodiment, the formulation comprises 0.01% to 0.05% polysorbate 80.In an embodiment, the formulation comprises 0.02% polysorbate 80. Forexample, formulations F1, F4, F9, F11, F13, F15, F17, F19, and F21contain polysorbate 80.

In an embodiment, the formulation comprises 10 mg/mL to 50 mg/mL of theanti-dengue antibody molecule, 10 mM to 50 mM histidine, 50 mM to 150 mMsodium chloride, and 50 mM to 150 mM sucrose, wherein the formulationhas a pH of 6 to 7. In an embodiment, the formulation comprises 25 mg/mLof the anti-dengue antibody molecule, 25 mM histidine, 75 mM sodiumchloride, and 100 mM sucrose, wherein the formulation has a pH of 6.5.

In an embodiment, the formulation comprises 10 mg/mL to 50 mg/mL of theanti-dengue antibody molecule, 10 mM to 50 mM histidine, 50 mM to 150 mMsodium chloride, 50 mM to 150 mM sucrose, 0.01% to 0.05% polysorbate 80,wherein the formulation has a pH of 6 to 7. In an embodiment, theformulation comprises 25 mg/mL of the anti-dengue antibody molecule, 25mM histidine, 75 mM sodium chloride, 100 mM sucrose, 0.02% polysorbate80, wherein the formulation has a pH of 6.5.

In an embodiment, the formulation comprises 10 mg/mL to 50 mg/mL of theanti-dengue antibody molecule, 10 mM to 50 mM histidine, 50 mM to 150 mMsodium chloride, and 50 mM to 150 mM arginine, wherein the formulationhas a pH of 6 to 7. In an embodiment, the formulation comprises about 25mg/mL of the anti-dengue antibody molecule, 25 mM histidine, 75 mMsodium chloride, and 75 mM arginine, wherein the formulation has a pH of6.5.

In an embodiment, the formulation comprises 10 mg/mL to 50 mg/mL of theanti-dengue antibody molecule, 10 mM to 50 mM histidine, 50 mM to 150 mMsodium chloride, 50 mM to 150 mM arginine, 0.01% to 0.05% polysorbate80, wherein the formulation has a pH of 6 to 7. In an embodiment, theformulation comprises 25 mg/mL of the anti-dengue antibody molecule, 25mM histidine, 75 mM sodium chloride, 75 mM arginine, 0.02% polysorbate80, wherein the formulation has a pH of 6.5.

In an embodiment, the formulation comprises 10 mg/mL to 50 mg/mL of theanti-dengue antibody molecule, 10 mM to 50 mM sodium citrate, 50 mM to150 mM sodium chloride, and 50 mM to 150 mM sucrose, wherein theformulation has a pH of 6 to 7. In an embodiment, the formulationcomprises 25 mg/mL of the anti-dengue antibody molecule, 25 mM sodiumcitrate, 75 mM sodium chloride, and 75 mM sucrose, wherein theformulation has a pH of 6.5.

In an embodiment, sodium citrate can be replaced with sodium phosphate,e.g., at an appropriate concentration and/or pH. For exampleformulations F6-F7 contain sodium phosphate. In an embodiment, sucrosecan be replaced with sorbitol, e.g., at an appropriate concentration.For example, formulations F16, F17, F20, and F21 contain sorbitol.

In an embodiment, the formulation is a liquid formulation.

In an embodiment, an anti-dengue antibody molecule formulation comprisesor consists of about 25 mg/mL of an anti-dengue antibody moleculedescribed herein (e.g., D88), about 25 mM histidine, about 75 mM sodiumchloride, and about 75 mM arginine, at pH 6.5. In an embodiment, ananti-dengue antibody molecule formulation comprises or consists of about25 mg/mL of an anti-dengue antibody molecule described herein (e.g.,D88), about 25 mM histidine, about 75 mM sodium chloride, about 75 mMarginine, and 0.02% polysorbate 80, at pH 6.5. In a further embodiment,an anti-dengue antibody molecule formulation comprises or consists ofabout 25 mg/mL of an anti-dengue antibody molecule described herein(e.g., D88), about 25 mM histidine, about 75 mM sodium chloride, andabout 100 mM sucrose, at pH 6.5. In a further embodiment, an anti-dengueantibody molecule formulation comprises or consists of about 25 mg/mL ofan anti-dengue antibody molecule described herein (e.g., D88), about 25mM histidine, about 75 mM sodium chloride, about 100 mM sucrose, andabout 0.02% polysorbate 80, at pH 6.5. In a another embodiment, ananti-dengue antibody molecule formulation comprises or consists of about25 mg/mL of an anti-dengue antibody molecule described herein (e.g.,D88), about 25 mM sodium citrate, about 75 mM sodium chloride, and about75 mM sucrose, at pH 6.5.

In some embodiments, the disclosure provides any of the formulationsdescribed in Table 7. In an embodiment, the formulation is F1 asdescribed in Table 7. In another embodiment, the formulation is F2 asdescribed in Table 7. In another embodiment, the formulation is F3 asdescribed in Table 7. In another embodiment, the formulation is F4 asdescribed in Table 7. In another embodiment, the formulation is F5 asdescribed in Table 7. In another embodiment, the formulation is F6 asdescribed in Table 7. In another embodiment, the formulation is F7 asdescribed in Table 7. In another embodiment, the formulation is F8 asdescribed in Table 7. In another embodiment, the formulation is F9 asdescribed in Table 7. In another embodiment, the formulation is F10 asdescribed in Table 7. In another embodiment, the formulation is F11 asdescribed in Table 7. In another embodiment, the formulation is F12 asdescribed in Table 7. In another embodiment, the formulation is F13 asdescribed in Table 7. In another embodiment, the formulation is F14 asdescribed in Table 7. In another embodiment, the formulation is F15 asdescribed in Table 7. In another embodiment, the formulation is F16 asdescribed in Table 7. In another embodiment, the formulation is F17 asdescribed in Table 7. In another embodiment, the formulation is F18 asdescribed in Table 7. In another embodiment, the formulation is F19 asdescribed in Table 7. In another embodiment, the formulation is F20 asdescribed in Table 7. In another embodiment, the formulation is F21 asdescribed in Table 7.

Storage and Preparation Methods

Liquid

In some cases, formulations containing antibodies are stored as liquid.Accordingly, it is desirable that the formulation be relatively stableunder such conditions, including, at 4° C. or in room temperature. Onemethod of determining the suitability of a formulation is to subject asample formulation to agitation or storage (e.g., at 4° C., 25° C., or45° C.) for a period of time (e.g., one week, two weeks, or four weeks),determining the amount of LMW species and/or HMW species that accumulateafter the agitation or storage and comparing it to the amount of LMWspecies or HMW species present in the sample prior to the agitation orstorage procedure. An increase in the LMW or HMW species indicatesdecreased stability.

Freezing

In some cases, formulations containing antibodies are frozen forstorage. Accordingly, it is desirable that the formulation be relativelystable under such conditions, including, under freeze-thaw cycles. Onemethod of determining the suitability of a formulation is to subject asample formulation to at least two, e.g., three, four, five, eight, ten,or more cycles of freezing (at, for example −20° C. or −80° C.) andthawing (for example by fast thaw in a 37° C. water bath or slow thaw at2°−8° C.), determining the amount of LMW species and/or HMW species thataccumulate after the freeze-thaw cycles and comparing it to the amountof LMW species or HMW species present in the sample prior to thefreeze-thaw procedure. An increase in the LMW or HMW species indicatesdecreased stability.

Lyophilization

Formulations can be stored after lyophilization. Therefore, testing aformulation for the stability of the protein component of theformulation after lyophilization is useful for determining thesuitability of a formulation. The method is similar to that described,supra, for freezing, except that the sample formulation is lyophilizedinstead of frozen, reconstituted to its original volume, and tested forthe presence of LMW species and/or HMW species. The lyophilized sampleformulation is compared to a corresponding sample formulation that wasnot lyophilized. An increase in LMW or HMW species in the lyophilizedsample compared to the corresponding sample indicates decreasedstability in the lyophilized sample.

In general, a lyophilization protocol includes loading a sample into alyophilizer, a pre-cooling period, freezing, vacuum initiation, rampingto the primary drying temperature, primary drying, ramping to thesecondary drying temperature, secondary drying, and stoppering thesample. Additional parameters that can be selected for a lyophilizationprotocol include vacuum (e.g., in microns) and condenser temperature.Suitable ramp rates for temperature are between about 0.1° C./min. to 2°C./min., for example 0.1° C./min. to 1.0° C./min., 0.1° C./min. to 0.5°C./min., 0.2° C./min. to 0.5° C./min., 0.1° C./min., 0.2° C./min., 0.3°C./min., 0.4° C./min., 0.5° C./min., 0.6° C./min., 0.7° C./min., 0.8°C./min., 0.9° C./min., and 1.0° C./min. Suitable shelf temperaturesduring freezing for a lyophilization cycle are generally from about −55°C. to −5° C., −25° C. to −5° C., −20° C. to −5° C., −15° C. to −5° C.,−10° C. to −5° C., −10° C., −11° C., −12° C., −13° C., −14° C., −15° C.,−16° C., −17° C., −18° C., −19° C., −20° C., −21° C., −22° C., −23° C.,−24° C., or −25° C. Shelf temperatures can be different for primarydrying and secondary drying, for example, primary drying can beperformed at a lower temperature than secondary drying. In anon-limiting example, primary drying can be executed at 0° C. andsecondary drying at 25° C.

In some cases, an annealing protocol is used during freezing and priorto vacuum initiation. In such cases, the annealing time must be selectedand the temperature is generally above the glass transition temperatureof the composition. In general, the annealing time is about 2 to 15hours, about 3 to 12 hours, about 2 to 10 hours, about 3 to 5 hours,about 3 to 4 hours, about 2 hours, about 3 hours, about 5 hours, about 8hours, about 10 hours, about 12 hours, or about 15 hours. Thetemperature for annealing is generally from about −35° C. to about −5°C., for example from about −25° C. to about −8° C., about −20° C. toabout −10° C., about −25° C., about −20° C., about −15° C., about 0° C.,or about −5° C. In some cases, the annealing temperature is generallyfrom −35° C. to 5° C., for example from 25° C. to −8° C., −20° C. to−10° C., −25° C., −20° C., −15° C., 0° C., or 5° C.

In general, a lyophilization cycle can run from 10 hours to 100 hours,e.g., 20 hours to 80 hours, 30 hours to 60 hours, 40 hours to 60 hours,45 hours to 50 hours, 50 hours to 65 hours.

Non-limiting examples of the temperature range for storage of anantibody formulation are about −20° C. to about 50° C., e.g., about −15°C. to about 30° C., about −15° C. to about 20° C., about 5° C. to about25° C., about 5° C. to about 20° C., about 5° C. to about 15° C., about2° C. to about 12° C., about 2° C. to about 10 C, about 2° C. to about8° C., about 2° C. to about 6° C., 2 C, 3° C., 4° C., 5° C., 6° C., 7°C., 8° C., 10° C., 15° C., or 25° C. Notwithstanding the storagetemperatures, in certain cases, samples are stable under temperaturechanges that may transiently occur during storage and transportationconditions that can be anticipated for such compositions.

Spray-Drying

In some cases, a formulation is spray-dried and then stored.Spray-drying is conducted using methods known in the art, and can bemodified to use liquid or frozen spray-drying (e.g., using methods suchas those from Niro Inc. (Madison, Wis.), Upperton Particle Technologies(Nottingham, England), or Buchi (Brinkman Instruments Inc., Westbury,N.Y.), or U.S. Application Pulication Nos. 2003/0072718 and2003/0082276).

Determination of Antibody Molecule Integrity

The accumulation of LMW species and HMW species are useful measures ofantibody stability. Accumulation of either LMW or HMW in a formulationis indicative of instability of a protein stored as part of theformulation. Size exclusion chromatography with HPLC can be used todetermine the presence of LMW and HMW species. Suitable systems for suchmeasurements are known in the art, e.g., HPLC systems (Waters, Milford,Mass.). Other systems known in the art can be used to evaluate theintegrity of antibody in a formulation, for example, SDS-PAGE (tomonitor HMW and LMW species), bioassays of antibody activity,enzyme-linked immunosorbent assay, ability to bind purified targetprotein (e.g., dengue), and cation exchange-HPLC (CEX-HPLC; to detectvariants and monitor surface charge). In one example, a bioassay is acell-based assay in which inhibition of an dengue-dependent activity isexamined in the presence of different concentrations of formulatedantibodymolecule to demonstrate biological activity.

Articles of Manufacture

The present application also provides an article of manufacture thatincludes a formulation as described herein and provides instructions foruse of the formulation. The article of manufacture can include acontainer suitable for containing the formulation. A suitable containercan be, without limitation, a bottle, vial, syringe, test tube,nebulizer (e.g., ultrasonic or vibrating mesh nebulizers), i.v. solutionbag, or inhaler (e.g., a metered dose inhaler (MDI) or dry powderinhaler (DPI)). The container can be formed of any suitable materialsuch as glass, metal, or a plastic such as polycarbonate, polystyrene,or polypropylene. In general, the container is of a material that doesnot adsorb significant amounts of protein from the formulation and isnot reactive with components of the formulation. In some embodiments,the container is a clear glass vial with a West 4432/50 1319 siliconizedgray stopper or a West 4023 Durafluor stopper. In some embodiments, thecontainer is a syringe. In specific embodiments, the formulationcomprises about 25 mg/mL of an antibody molecule described herein, about25 mM histidine, about 75 mM sodium chloride, and about 100 mM sucrose,at a pH of about 6.5, in a pre-filled syringe. In certain embodiments,the syringe is suitable for use with an autoinjector device.

Examples of nebulizers include, in non-limiting examples, jetnebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. Theseclasses use different methods to create an aerosol from a liquid. Ingeneral, any aerosol-generating device that can maintain the integrityof the protein in these formulations is suitable for delivery offormulations as described herein.

Formulations to be used for administration to a subject, e.g., as apharmaceutical, must be sterile. This is accomplished using methodsknown in the art, e.g., by filtration through sterile filtrationmembranes, prior to, or following, formulation of a liquid orlyophilization and reconstitution. Alternatively, when it will notdamage structure, components of the formulation can be sterilized byautoclaving and then combined with filter or radiation sterilizedcomponents to produce the formulation.

Animal Models

The antibody molecules and formulations described herein can beevaluated in an animal model. For example, an animal model can be usedto test the efficacy of an antibody molecule or formulation describedherein in reducing dengue viral infection, replication and/ortransmission. Exemplary animal models that can be used for evaluating anantibody molecule or formulation described herein include, but are notlimited to, AG129 mouse models (e.g., as described in Tharakaraman etal., Proc Natl Acad Sci USA. 2013; 110(17):E1555-64; Johnson et al. JVirol. 1999; 73(1):783-6); non-mouse adapted mouse models (e.g.,non-mouse adapted DENV-2 D2Y98P mouse model as described in Tan et al.PLoS Negl Trop Dis. 2010; 4(4):e672); humanized mouse models (e.g., asdescribed in Sridharan et al. J Virol. 2013; 87(21):11648-58); non-humanprimate models (e.g., as described in Goncalvez et al. Proc Natl AcadSci USA. 2007; 104(22):9422-7); and mosquito models (e.g., as describedin Vu et al. PLoS Negl Trop Dis. 2010; 4(7):e757).

The AG129 mouse strain, which lacks both type-I and type-II interferonreceptors, is an animal model that replicates certain diseasemanifestations observed in clinical cases of dengue, including viremiaand other signs of disease (Tharakaraman et al., Proc Natl Acad Sci USA.2013; 110(17):E1555-64; Johnson et al. J Virol. 1999; 73(1):783-6). Thismodel is useful in evaluation of antiviral treatments and can also beused in proof of principle studies. Briefly, the AG129 (which isdeficient in IFN-α/β and IFN-γ receptors) mouse is challenged withdengue virus, and a candidate therapeutic antibody molecule isadministered. Typically, viremia (virus titer in a blood sample) is theendpoint of the experiment. Viremia can be measured, e.g., withquantitative RT-PCR. An exemplary AG129 mouse model is described inExample 10 of International Application Publication No. WO2015/122995.

Non-mouse adapted mouse models can be generated, e.g., using a non-mouseadapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129mice upon intraperitoneal administration (Tan et al. PLoS Negl Trop Dis.2010; 4(4):e672). Infection with a high dose of D2Y98P can inducecytokine storm, massive organ damage, and severe vascular leakage,leading to haemorrhage and rapid death of the animals at the peak ofviremia. Infection with a low dose of D2Y98P can lead to asymptomaticviral dissemination and replication in relevant organs, followed bynon-paralytic death of the animals few days after virus clearance,similar to the disease kinetic in humans. Spleen damage, liverdysfunction and increased vascular permeability, but no hemorrhage, canbe observed in moribund animals, suggesting intact vascular integrity, acardinal feature in dengue shock syndrome.

Humanized mouse models can be generated, e.g., by adoptive transfer ofhuman CD34⁺ fetal liver cells into NOD-scid Il2rg^(−/−) (NSG) mice thatdevelop significant levels of human platelets, monocytes/macrophages,and hepatocytes (Sridharan et al. J Virol. 2013; 87(21):11648-58).Infection of these mice with dengue virus such as DENV serotype 2(DENV-2) can recapture certain characteristic features of dengue viralinfection in humans, e.g., transient leukopenia and thrombocytopenia.

Non-human primate models can be generated, e.g., in juvenile rhesusmonkeys after DENV challenge, as described in Goncalvez et al. Proc NatlAcad Sci USA. 2007; 104(22):9422-7. The viremia titers of infectedmonkeys can be determined, e.g., by quantitative PCR or Focus FormingUnits (FFU) assay.

Mosquito models can also be used to evaluate inhibitory activity ofantibodies against dengue virus, e.g., neutralization of viral infectionor reduction of transmission between infected subjects and mosquitoes.Dengue virus is a mosquito transmitted RNA virus. Certain dengue viruscan develop in vivo fitness advantage, which may result in higherprobability of human-to-mosquito transmission (Vu et al., PLoS Negl TropDis. 2010; 4(7):e757). To establish a mosquito model, blood containingvirus and antibody can be fed to mosquitoes. Viral load in mosquitoes'abdomens can be measured by qRT-PCR. An exemplary mosquito model isdescribed in Example 13 of International Application Publication No.WO2015/122995.

Pharmaceutical Compositions and Kits

In some aspects, this disclosure provides compositions, e.g.,pharmaceutically acceptable compositions, which include an anti-dengueantibody molecule described herein, formulated together with apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier can be suitable for intravenous,intramuscular, subcutaneous, parenteral, rectal, spinal or epidermaladministration (e.g., by injection or infusion). In certain embodiments,less than about 5%, e.g., less than about 4%, 3%, 2%, or 1% of theantibody molecules in the pharmaceutical composition are present asaggregates. In other embodiments, at least about 95%, e.g., at leastabout 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%, or more of the antibodymolecules in the pharmaceutical composition are present as monomers. Insome embodiments, the level of antibody aggregates or monomers isdetermined by chromatography, e.g., high performance size exclusionchromatography (HP-SEC).

The compositions set out herein may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes, and suppositories. A suitable form depends onthe intended mode of administration and therapeutic application. Typicalsuitable compositions are in the form of injectable or infusiblesolutions. One suitable mode of administration is parenteral (e.g.,intravenous, subcutaneous, intraperitoneal, intramuscular). In someembodiments, the antibody molecule is administered by intravenousinfusion or injection. In certain embodiments, the antibody isadministered by intramuscular or subcutaneous injection.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high antibody concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The formulations, e.g., pharmaceutical formulations or compositions,comprising the antibody molecules described herein can be administeredby a variety of methods. Several are known in the art, and for manytherapeutic applications, an appropriate route/mode of administration isintravenous injection or infusion. For example, the antibody moleculescan be administered by intravenous infusion at a rate of less than 10mg/min; preferably less than or equal to 5 mg/min to reach a dose ofabout 1 to 100 mg/m², preferably about 5 to 50 mg/m², about 7 to 25mg/m² and more preferably, about 10 mg/m². As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the activecompound may be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antibody molecule can be orally administered,for example, with an inert diluent or an assimilable edible carrier. Theantibody molecule (and other ingredients, if desired) may also beenclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the antibody molecule may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. To administer an antibody molecule by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.Therapeutic compositions can also be administered with medical devices,and several are known in the art.

Dosage regimens are adjusted to provide the desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms are dictated by and directly dependent on (a)the unique characteristics of the antibody molecule and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an antibody molecule for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody molecule is 0.1-20mg/kg, more preferably 1-10 mg/kg. The antibody molecule can beadministered by intravenous infusion at a rate of less than 10 mg/min,preferably less than or equal to 5 mg/min to reach a dose of about 1 to100 mg/m², preferably about 5 to 50 mg/m², about 7 to 25 mg/m², and morepreferably, about 10 mg/m². It is to be noted that dosage values mayvary with the type and severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that dosageranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed compositions.

The pharmaceutical formulations or compositions herein may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody molecule. A “therapeutically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic result. A therapeuticallyeffective amount of the modified antibody or antibody fragment may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the antibody or antibody portion toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody molecule is outweighed by the therapeutically beneficialeffects. A “therapeutically effective dosage” preferably inhibits ameasurable parameter by at least about 20%, more preferably by at leastabout 40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. Themeasurable parameter may be, e.g., viral load, fever, headache, muscleor joint pains, skin rash, bleeding, reduced platelet levels, andreduced blood pressure. The ability of an antibody molecule to inhibit ameasurable parameter can be evaluated in an animal model systempredictive of efficacy in dengue fever. Alternatively, this property ofa composition can be evaluated by examining the ability of the antibodymolecule to neutralize dengue virus, e.g., by assaying focus formationin vitro.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Also within this disclosure is a kit comprising a formulation comprisingan antibody molecule described herein. The kit can include one or moreother elements including: instructions for use; other reagents, e.g., alabel, a therapeutic agent, or an agent useful for chelating, orotherwise coupling, an antibody to a label or therapeutic agent, or aradioprotective composition; devices or other materials for preparingthe antibody molecule for administration; pharmaceutically acceptablecarriers; and devices or other materials for administration to asubject.

Nucleic Acids

The present disclosure also features formulations of anti-dengueantibody molecules encoded by nucleic acids comprising nucleotidesequences that encode heavy and light chain variable regions and CDRs ofthe anti-dengue antibody molecules, as described herein. For example,the present disclosure features a first and second nucleic acid encodingheavy and light chain variable regions, respectively, of an anti-dengueantibody molecule chosen from one or more of the antibody moleculesdisclosed herein, e.g., an antibody of Table 1, or a portion of anantibody, e.g., the variable regions of Table 2. The nucleic acid cancomprise a nucleotide sequence encoding any one of the amino acidsequences in the tables herein, or a sequence substantially identicalthereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, or which differs by no more than 3, 6, 15, 30, or 45nucleotides from the sequences shown in the tables herein.

In certain embodiments, the nucleic acid can comprise a nucleotidesequence encoding at least one, two, or three CDRs from a heavy chainvariable region having an amino acid sequence as set forth in the tablesherein, or a sequence substantially homologous thereto (e.g., a sequenceat least about 85%, 90%, 95%, 99% or more identical thereto, and/orhaving one or more substitutions, e.g., conserved substitutions). Insome embodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, or three CDRs from a light chain variableregion having an amino acid sequence as set forth in the tables herein,or a sequence substantially homologous thereto (e.g., a sequence atleast about 85%, 90%, 95%, 99% or more identical thereto, and/or havingone or more substitutions, e.g., conserved substitutions). In someembodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, three, four, five, or six CDRs from heavyand light chain variable regions having an amino acid sequence as setforth in the tables herein, or a sequence substantially homologousthereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, and/or having one or more substitutions, e.g.,conserved substitutions).

In certain embodiments, the nucleic acid can comprise a nucleotidesequence encoding at least one, two, or three CDRs from a heavy chainvariable region having the nucleotide sequence as set forth in Table 5herein, a sequence substantially homologous thereto (e.g., a sequence atleast about 85%, 90%, 95%, 99% or more identical thereto, and/or capableof hybridizing under the stringency conditions described herein). Insome embodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, or three CDRs from a light chain variableregion having the nucleotide sequence as set forth in Table 5 herein, ora sequence substantially homologous thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or capable ofhybridizing under the stringency conditions described herein). Incertain embodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, three, four, five, or six CDRs from heavyand light chain variable regions having the nucleotide sequence as setforth in Table 5 herein, or a sequence substantially homologous thereto(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, and/or capable of hybridizing under the stringency conditionsdescribed herein).

In certain embodiments, the nucleic acid comprises a nucleotide sequenceas set forth in Table 5 herein or a sequence substantially homologousthereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, and/or capable of hybridizing under the stringencyconditions described herein). In some embodiments, the nucleic acidcomprises a portion of a nucleotide sequence as set forth in Table 5herein or a sequence substantially homologous thereto (e.g., a sequenceat least about 85%, 90%, 95%, 99% or more identical thereto, and/orcapable of hybridizing under the stringency conditions describedherein). The portion may encode, for example, a variable region (e.g.,VH or VL); one, two, or three or more CDRs; or one, two, three, or fouror more framework regions.

The nucleic acids disclosed herein include deoxyribonucleotides orribonucleotides, or analogs thereof. The polynucleotide may be eithersingle-stranded or double-stranded, and if single-stranded may be thecoding strand or non-coding (antisense) strand. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.The nucleic acid may be a recombinant polynucleotide, or apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich either does not occur in nature or is linked to anotherpolynucleotide in a nonnatural arrangement.

In some aspects, the application features host cells and vectorscontaining the nucleic acids described herein. The nucleic acids may bepresent in a single vector or separate vectors present in the same hostcell or separate host cell, as described in more detail herein below.

Vectors

Further provided herein formulations of anti-dengue antibody moleculesproduced from vectors comprising nucleotide sequences encoding anantibody molecule described herein. In some embodiments, the vectorscomprise nucleotides encoding an antibody molecule described herein. Insome embodiments, the vectors comprise the nucleotide sequencesdescribed herein. The vectors include, but are not limited to, a virus,plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC).

Numerous vector systems can be employed. For example, one class ofvectors utilizes DNA elements which are derived from animal viruses suchas, for example, bovine papilloma virus, polyoma virus, adenovirus,vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV orMOMLV) or SV40 virus. Another class of vectors utilizes RNA elementsderived from RNA viruses such as Semliki Forest virus, Eastern EquineEncephalitis virus and Flaviviruses.

Additionally, cells which have stably integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow for the selection of transfected host cells. The marker mayprovide, for example, prototropy to an auxotrophic host, biocideresistance, (e.g., antibiotics), or resistance to heavy metals such ascopper, or the like. The selectable marker gene can be either directlylinked to the DNA sequences to be expressed, or introduced into the samecell by cotransformation. Additional elements may also be needed foroptimal synthesis of mRNA. These elements may include splice signals, aswell as transcriptional promoters, enhancers, and termination signals.

Once the expression vector or DNA sequence containing the constructs hasbeen prepared for expression, the expression vectors may be transfectedor introduced into an appropriate host cell. Various techniques may beemployed to achieve this, such as, for example, protoplast fusion,calcium phosphate precipitation, electroporation, retroviraltransduction, viral transfection, gene gun, lipid based transfection orother conventional techniques. In the case of protoplast fusion, thecells are grown in media and screened for the appropriate activity.

Methods and conditions for culturing the resulting transfected cells andfor recovering the antibody molecule produced are known to those skilledin the art, and may be varied or optimized depending upon the specificexpression vector and mammalian host cell employed, based upon thepresent description.

Cells

The present disclosure also provides formulations of anti-dengueantibody molecules produced from host cells comprising a nucleic acidencoding an antibody molecule as described herein. For example, the hostcells may comprise a nucleic acid of Table 5, a sequence substantiallyhomologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99%or more identical thereto, and/or capable of hybridizing under thestringency conditions described herein), or a portion of one of saidnucleic acids. Additionally, the host cells may comprise a nucleic acidencoding an amino acid sequence of Table 2 or Table 3, a sequencesubstantially homologous thereto (e.g., a sequence at least about 85%,90%, 95%, 99% or more identical thereto), or a portion of one of saidsequences.

In some embodiments, the host cells are genetically engineered tocomprise nucleic acids encoding the antibody molecule.

In certain embodiments, the host cells are genetically engineered byusing an expression cassette. The phrase “expression cassette,” refersto nucleotide sequences, which are capable of affecting expression of agene in hosts compatible with such sequences. Such cassettes may includea promoter, an open reading frame with or without introns, and atermination signal. Additional factors necessary or helpful in effectingexpression may also be used, such as, for example, an induciblepromoter.

The disclosure also provides host cells comprising the vectors describedherein.

The cell can be, but is not limited to, a eukaryotic cell, a bacterialcell, an insect cell, or a human cell. Suitable eukaryotic cellsinclude, but are not limited to, Vero cells, HeLa cells, COS cells, CHOcells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cellsinclude, but are not limited to, Sf9 cells.

Uses of Formulations of Anti-Dengue Antibody Molecules

The formulations comprising the antibody molecules disclosed herein havein vitro and in vivo pharmaceutical utilities (e.g., therapeutic andprophylactic, and/or diagnostic utilities). In some embodiments, theantibody molecules neutralize dengue virus. For example, these moleculescan be administered to cells in culture, in vitro or ex vivo, or to asubject, e.g., a human subject, e.g., in vivo, to neutralize denguevirus. Accordingly, in some aspects, the disclosure provides a method oftreating a dengue virus infection in a subject, comprising administeringto the subject a formulation as described herein of an antibody moleculedescribed herein, such that the dengue virus infection is treated. Forexample, these formulations can be administered to cells in culture,e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat,prevent, and/or diagnose a dengue virus infection.

As used herein, the term “subject” is intended to include human andnon-human animals. In some embodiments, the subject is a human subject,e.g., a human patient infected with dengue virus or at risk of beinginfected with dengue virus. The term “non-human animals” includesmammals and non-mammals, such as non-human primates. In someembodiments, the subject is a human. The methods and compositionsdescribed herein are suitable for treating human patients infected withdengue virus. Patients infected with dengue virus include those who havebeen exposed to the virus but are (at least temporarily) asymptomatic,patients having dengue fever, patients having dengue hemorrhagic fever,and patients having dengue shock syndrome.

Methods of Treating Dengue Virus

Dengue virus displays an E (envelope) protein on the viral surface. TheE protein contributes to the attachment of the virus to a host cell. TheE protein comprises a DI domain (a nine-stranded beta-barrel) a DIIdomain (a hydrophobic domain implicated in fusion with the host cell),and a DIII domain (an extracellular domain). While not wishing to bebound by theory, in some embodiments, the antibody molecules describedherein can neutralize dengue virus by binding to its E protein DIII(EDIII) domain, e.g., by preventing the virus from fusing with a hostcell, preventing the virus from binding to a host cell, disrupting thestructure of the E protein, or destabilizing the virus.

Dengue fever is an infectious disease, usually mosquito-borne, caused bythe dengue virus. The initial infection is often followed by a briefasymptomatic period, usually 4-7 days. Sometimes an infected patientdoes not develop any symptoms of dengue fever. However, in patients thatmanifest dengue fever, the characteristic symptoms are sudden-onsetfever (sometimes over 40° C.), headache, muscle and joint pains, andrash. During the febrile phase of infection, fever, pain, and headachemanifest. In some patients the febrile phase is followed by the criticalphase (associated with dengue shock syndrome and dengue hemorrhagicfever), in which patients may suffer from fluid accumulation in thechest and abdominal cavity, depletion of fluid from circulation, aninadequate supply of blood to the vital organs, and bleeding. This isfollowed by a recovery phase. In some embodiments, the antibodymolecules herein are administered to a patient in the asymptomaticperiod, the febrile phase, the critical phase, and/or the recoveryphase.

Dengue virus is typically diagnosed based on a physical exam and thepatient's reported symptoms. A probable diagnosis can be made when apatient displays a fever and at least two symptoms selected fromnausea/vomiting, rash, generalized pain, reduced white blood celllevels, or positive tourniquet test. Additional tests that indicatedengue fever include a test for reduced white blood cell count, lowplatelet levels, metabolic acidosis, elevated level of aminotransferasefrom the liver, hemoconcentration, hypoalbuminemia, detection of fluidby ultrasound (suggests dengue shock syndrome), a pulse pressure below20 mm Hg (indicates dengue shock syndrome), delayed capillary refill(indicates peripheral vascular collapse). Accordingly, in someembodiments the antibody molecules are administered to a patient thatsatisfies the aforementioned criteria.

Certain antibody molecules described herein are capable of treating atleast two, three, or four serotypes of dengue virus. Accordingly, incertain embodiments, the antibody molecule is administered to a patientinfected with or with a risk of being infected with dengue virus, whenno test has been performed to determine the serotype of the denguevirus, e.g., the serotype of the dengue virus may be unknown. In someembodiments, the dengue virus is of serotype DV-1, DV-2, DV-3, or DV-4.

The antibody molecules are typically administered at a frequency thatkeeps a therapeutically effective level of antibodies in the patient'ssystem until the patient recovers. For example, the antibody moleculesmay be administered at a frequency that achieves a serum concentrationsufficient for at least about 1, 2, 5, 10, 20, 30, or 40 antibodies tobind each virion. In some embodiments, the antibody molecules areadministered every 1, 2, 3, 4, 5, 6, or 7 days.

Methods of administering various formulations and antibody molecules areknown in the art and are described below. Suitable dosages of theantibody molecules used will depend on the age and weight of the subjectand the particular drug used.

The antibody molecules can be used by themselves or conjugated to asecond agent, e.g., an antiviral agent, toxin, or protein, e.g., asecond anti-dengue antibody. This method includes: administering theantibody molecule, alone or conjugated to a second agent, to a subjectrequiring such treatment. The antibody molecules can be used to delivera variety of therapeutic agents, e.g., a toxin or anti-viral agent, ormixtures thereof.

Combination Therapies

Formulations including the anti-dengue antibody molecules can be used incombination with other therapies. For example, the combination therapycan include an anti-dengue antibody molecule co-formulated with, and/orco-administered with, one or more additional therapeutic agents, e.g.,anti-viral agents (including other anti-dengue antibodies), vaccines(including dengue virus vaccines), or agents that enhance an immuneresponse. In other embodiments, the antibody molecules are administeredin combination with other therapeutic treatment modalities, such asintravenous hydration, fever-reducing agents (such as acetaminophen), orblood transfusion. Such combination therapies may advantageously utilizelower dosages of the administered therapeutic agents, thus avoidingpossible toxicities or complications associated with the variousmonotherapies.

Administered “in combination”, as used herein, means that two (or more)different treatments are delivered to the subject before, or during thecourse of the subject's affliction with the disease. In one embodiment,two or more treatments are delivered prophylactically, e.g., before thesubject is infected or diagnosed with dengue virus. In anotherembodiment, the two or more treatments are delivered after the subjecthas been diagnosed with the dengue virus. In some embodiments, thedelivery of one treatment is still occurring when the delivery of thesecond begins, so that there is overlap. This is sometimes referred toherein as “simultaneous” or “concurrent delivery.” In other embodiments,the delivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

The anti-viral agent may be, e.g., balapiravir, chloroquine, celgosivir,ivermectin, or Carica folia.

The vaccine may be, e.g., live, attenuated, recombinant dengue serotypes1, 2, 3, and 4 virus (e.g., clinical trial NCT01488890 by SanofiPasteur); CYD Tetravalent Dengue Vaccine (e.g., clinical trialNCT01943825, by Sanofi Pasteur), Chimeric dengue serotype (1, 2, 3, 4)(e.g., clinical trial NCT00730288 by Sanofi), CYD Dengue Vaccine (e.g.,clinical trial NCT00993447 by Sanofi), tetravalent live attenuateddengue vaccine (e.g., clinical trial NCT00322049 by GlaxoSmithKline),Tetravalent Dengue Vaccine (TVDV) (e.g., clinical trial NCT01502358 byU.S. Army Medical Research and Materiel Command), Chimeric tetravalentdengue (serotype 1, 2, 3, 4) (e.g., clinical trial NCT00842530 by SanofiPasteur), dengue lyophilized vaccine (e.g., clinical trial NCT01696422by Butantan Institute), ChimeriVax™ Tetravalent Dengue Vaccine (e.g.,clinical trial NCT00617344 by Sanofi), Bivalent CYD-1,3 Dengue (Vero)(e.g., clinical trial NCT00740155 by Sanofi Pasteur), Bivalent CYD-2,4Dengue (Vero) (e.g., clinical trial NCT00740155 by Sanofi Pasteur),Tetravalent blending VDV-2/CYD-1,3,4 Dengue (Vero) (e.g., clinical trialNCT00740155 by Sanofi Pasteur), Tetravalent CYD-1,2,3,4 Dengue (Vero)(e.g., clinical trial NCT00740155 by Sanofi Pasteur), rDEN1delta30 orrDEN2/4delta30(ME) (e.g., clinical trial NCT00458120 by NationalInstitute of Allergy and Infectious Diseases), Modified Live TetravalentChimeric Dengue Vaccine (SC or ID) (e.g., clinical trial NCT01110551 byNational Institute of Allergy and Infectious Diseases), Dengue vaccine(e.g., clinical trial NCT00384670 by United States Army Medical MaterielDevelopment Activity), Investigational Vaccine for Dengue Virus Subtype2 (e.g., NCT01073306 by National Institute of Allergy and InfectiousDiseases), F 17 (e.g., NCT01843621 by U.S. Army Medical Research andMateriel Command), Post-Transfection F17 or Post-Transfection F19 (e.g.,clinical trial NCT00468858 by U.S. Army Medical Research and MaterielCommand), DENVax (e.g., clinical trial NCT01511250 by Inviragen Inc.),DIME (dengue-1 premembrane/envelope DNA vaccine) (e.g., clinical trialNCT00290147 by U.S. Army Office of the Surgeon General), InvestigationalVaccine for DEN1 (e.g., clinical trial NCT01084291 by National Instituteof Allergy and Infectious Diseases), Live attenuated tetravalent denguevaccine (e.g., clinical trial NCT00350337 by Walter Reed Army Instituteof Research), rDEN4delta30-200,201 (e.g., clinical trial NCT00270699 byNational Institute of Allergy and Infectious Diseases),TetraVax-DV-TV003 or rDEN2430-7169 (e.g., clinical trial NCT02021968 byNational Institute of Allergy and Infectious Diseases), TetraVax-DV,optionally in admixture (e.g., clinical trial NCT01436422 by NationalInstitute of Allergy and Infectious Diseases), DEN4 Vaccine Candidate(e.g., clinical trial NCT00919178 by National Institute of Allergy andInfectious Diseases), rDEN4delta30-4995 (e.g., clinical trialNCT00322946 by National Institute of Allergy and Infectious Diseases),rDEN3delta30/31-7164 (e.g., clinical trial NCT00831012 by NationalInstitute of Allergy and Infectious Diseases), TDENV-PIV (e.g., clinicaltrial NCT01702857 by U.S. Army Medical Research and Materiel Command),DENV-1 PIV (e.g., clinical trial NCT01502735 by U.S. Army MedicalResearch and Materiel Command), rDEN3-3′D4delta30 (e.g., clinical trialNCT00712803 by National Institute of Allergy and Infectious Diseases),V180 (e.g., clinical trial NCT01477580 by Merck Sharp & Dohme Corp.), orDEN1-80E (e.g., clinical trial NCT00936429 by Hawaii Biotech, Inc.).

The other therapy may be, for example, hypertonic sodium lactate,activated recombinant human factor VII, or anti-d (e.g., clinical trialNCT01443247 by Postgraduate Institute of Medical Education andResearch).

In certain embodiments, the additional antiviral agent is a secondanti-dengue antibody molecule, e.g., an anti-dengue antibody moleculedifferent from a first anti-dengue antibody molecule. Exemplaryanti-dengue antibody molecules that can be used in combination include,but are not limited to, any combination of the antibodies listed inTable 1 (for example, any combination of two of more of D88, F38, A48,C88, F108, B48, A68, A100, C58, C78, C68, D98, A11 (also known asmonoclonal antibody 4E5A (Tharakaraman et al., Proc Natl Acad Sci USA.2013; 110(17):E1555-64)) or B11; monoclonal antibody 4E11 (Thullier etal., J Biotechnol. 1999; 69(2-3):183-90); human antibody 14c10 (HM14c10)(Teoh et al. Sci Transl Med. 2012 Jun. 20; 4(139):139ra83); humanmonoclonal antibodies 1F4, 2D22, and 5J7 (de Alwis et al., Proc NatlAcad Sci USA. 2012; 109(19):7439-44); human monoclonal antibodies DV1.1,DV1.6, DV3.7, DV4.4, DV5.1, DV6.1, DV7.5, DV8.1, DV10.16, DV13.4,DV13.8, DV14.5, DV14.5, DV15.7, DV16.5, DV16.8, DV17.6, DV18.21, DV18.4,DV19.3, DV20.1, DV21.1, DV21.5, DV22.3, DV22.3 LALA, DV23.13, DV25.5,DV27.2, DV28.1, DV28.8, DV34.4, DV35.3, DV38.1, DV51.6, DV52.1, DV53.4,DV54.7, DV55.1, DV56.12, DV54.7, DV57.4, DV59.3, DV60.3, DV61.2, DV62.5,DV63.1, DV64.3, DV65.5, DV66.1, DV67.9, DV68.2, DV69.6, DV70.1, DV71.1,DV74.4, DV75.9, DV76.5, DV77.5, DV78.6, DV79.3, DV82.11, DV82.11 LALA,DV86.2, DV87.1, DV87.1 LALA, DV90.3, DV257.13, DV291.7, DV415.8, andDV470.12 (Beltramello et al., Cell Host Microbe. 2010; 8(3):271-83);human monoclonal antibodies 3-147, 58/5, 2F5, 2G4, 5F9, and 135.3(Dejnirattisai et al., Science. 2010; 328(5979):745-8); mAb 2H12(Midgley et al. J Immunol. 2012; 188(10):4971-9); humanized monoclonalantibody 1A5 (Goncalvez et al., Proc Natl Acad Sci USA. 2007;104(22):9422-7); and human monoclonal antibody 1C19 (Smith et al., MBio.2013; 4(6):e00873-13); or any of the antibodies disclosed in: WO05/056600 by Lai, C. and Purcell, R. (e.g., antibodies 1A5 and 5H2;WO2010/043977 by Lanzavecchia, A. et al.; WO2013/173348 by Dimitrov etal.; US2013/0259871 by Macary et al.; WO 2013/089647 by Fink et al.; WO2013/035345 by Setthapramote et al.; U.S. Pat. No. 8,637,035 byHan-Chung Wu et al.; or WO 2014/025546 by Sasisekharan, R. et al.; or aderivative of any of the aforesaid antibodies (e.g., a human orhumanized form thereof).

Other therapeutic agents that can be used in combination with ananti-dengue antibody described herein also include, but are not limitedto, for example, alpha-glucosidase I inhibitors (e.g., celgosivir asdescribed in Rathore et al., Antiviral Res. 2011; 92(3):453-60);adenosine nucleoside inhibitors (e.g., NITD008 as described in Yin etal., Proc Natl Acad Sci USA. 2009; 106(48):20435-9); inhibitors of NS3and/or its cofactor NS2B (e.g., compounds that block the NS2B bindingpocket within NS3, e.g., [5-amino-1-(phenyl)sulfonyl-pyrazol-3-yl]compounds, as described in Lescar et al., Antiviral Res. 2008;80(2):94-101); RNA-dependent RNA polymerase (RdRp) inhibitors (e.g.,NITD107 as described in Noble et al., J Virol. 2013; 87(9):5291-5);inhibitors of host pyrimidine biosynthesis, e.g., host dihydroorotatedehydrogenase (DHODH) (e.g., NITD-982 and brequinar as described in Wanget al., J Virol. 2011; 85(13):6548-56); inhibitors of viral NS4B protein(e.g., NITD-618 as described in Xie et al., J Virol. 2011;85(21):11183-95); and iminosugars (e.g., UV-4 as described in Perry etal., Antiviral Res. 2013; 98(1):35-43).

Methods of Diagnosis

In some aspects, the present disclosure provides a diagnostic method fordetecting the presence of a dengue virus E protein in vitro (e.g., in abiological sample, such as a blood sample) or in vivo (e.g., in vivoimaging in a subject). The method includes: (i) contacting the samplewith an antibody molecule described herein, or administering to thesubject, the antibody molecule; (optionally) (ii) contacting a referencesample, e.g., a control sample (e.g., a control biological sample, suchas plasma or blood) or a control subject with an antibody moleculedescribed herein; and (iii) detecting formation of a complex between theantibody molecule, and the sample or subject, or the control sample orsubject, wherein a change, e.g., a statistically significant change, inthe formation of the complex in the sample or subject relative to thecontrol sample or subject is indicative of the presence of dengue virusin the sample. The antibody molecule can be directly or indirectlylabeled with a detectable substance to facilitate detection of the boundor unbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescent materialsand radioactive materials, as described above and described in moredetail below.

The term “sample,” as it refers to samples used for detectingpolypeptides includes, but is not limited to, cells, cell lysates,proteins or membrane extracts of cells, body fluids such as blood, ortissue samples.

Complex formation between the antibody molecule and a dengue virusprotein can be detected by measuring or visualizing either the antibodymolecule bound to the dengue virus protein or unbound antibody molecule.Any suitable detection assays can be used, and conventional detectionassays include an enzyme-linked immunosorbent assays (ELISA), aradioimmunoassay (RIA) or tissue immunohistochemistry. Alternative tolabeling the antibody molecule, the presence of a dengue virus proteincan be assayed in a sample by a competition immunoassay utilizingstandards labeled with a detectable substance and an unlabeled antibodymolecule. In this assay, the biological sample, the labeled standardsand the antibody molecule are combined and the amount of labeledstandard bound to the unlabeled binding molecule is determined. Theamount of a dengue virus protein in the sample is inversely proportionalto the amount of labeled standard bound to the antibody molecule.

EXAMPLES Example 1. Screening of Antibody Formulations

This Example summarizes a screening study for an exemplary anti-dengueantibody molecule described herein, e.g., D88. Suitable formulationcomponents and candidate formulations were tested.

Methods

The formulation components and candidate formulations used in the studyare shown in Table 7. All formulations contain 25 mg/mL of the D88anti-dengue antibody. The timepoint schedule is shown in Table 8.

TABLE 7 Formulation components and candidate formulations used for thestudy No. Formulation Components F1 10 mM sodium citrate pH 6.0, 100 mMNaCl, 0.02% polysorbate 80 F2 25 mM sodium citrate pH 6.5, 75 mM NaCl,75 mM arginine F3 25 mM sodium citrate pH 6.5, 150 mM arginine F4 25 mMsodium citrate pH 6.5, 150 mM arginine, 0.02% polysorbate 80 F5 25 mMsodium citrate pH 6.5, 75 mM NaCl, 75 mM sucrose F6 25 mM sodiumphosphate pH 7.0, 75 mM NaCl, 75 mM arginine F7 25 mM sodium phosphatepH 7.0, 75 mM NaCl, 75 mM sucrose F8 25 mM histidine pH 6.5, 150 mM NaClF9 25 mM histidine pH 6.5, 150 mM NaCl, 0.02% polysorbate 80 F10 25 mMhistidine pH 6.5, 150 mM arginine F11 25 mM histidine pH 6.5, 150 mMarginine, 0.02% polysorbate 80 F12 25 mM histidine pH 6.5, 75 mM NaCl,75 mM arginine F13 25 mM histidine pH 6.5, 75 mM NaCl, 75 mM arginine,0.02% polysorbate 80 F14 25 mM histidine pH 6.5, 75 mM NaCl, 100 mMsucrose F15 25 mM histidine pH 6.5, 75 mM NaCl, 100 mM sucrose, 0.02%polysorbate 80 F16 25 mM histidine pH 6.5, 75 mM NaCl, 125 mM sorbitolF17 25 mM histidine pH 6.5, 75 mM NaCl, 125 mM sorbitol, 0.02%polysorbate 80 F18 25 mM arginine pH 7.0, 75 mM NaCl, 100 mM sucrose F1925 mM arginine pH 7.0, 75 mM NaCl, 100 mM sucrose, 0.02% polysorbate 80F20 25 mM arginine pH 7.0, 75 mM NaCl, 125 mM sorbitol F21 25 mMarginine pH 7.0, 75 mM NaCl, 125 mM sorbitol, 0.02% polysorbate 80 Study10 mM sodium citrate pH 6.0, 100 mM NaCl, 0.02% polysorbate 80 Control

TABLE 8 Timepoint schedule used for the study Timepoint (Months)Temperature 0 1  +5° C. X{circumflex over ( )}⁺* X{circumflex over ( )}+40° C. X X = Vial taken for assay. {circumflex over ( )}= Includes MFItesting. ⁺= Freeze-thaw at −65° C. & below performed on all formulationsat T = 0 (3 cycles). *= Agitation performed on all formulations at T = 0(200 rpm on horizontal plate shaker for 24 hrs at ambient temperature).

Samples of the formulation were analyzed at two time points, at time 0and one month.

Results

Twenty-one candidate formulations were prepared and analyzed, along witha study control (10 mM sodium citrate pH 6.0, 100 mM NaCl, 0.02%polysorbate 80). The osmolality of the formulations is shown in FIG. 2A.The pH of the formulations is shown in FIG. 2B.

The formulations were analyzed according to visual appearance, gelpermeation high performance liquid chromatography, reduced SDS PAGE,non-reduced SDS PAGE, isoelectric focusing, optical density, dynamiclight scattering, differential scanning calorimetry, and sub-visibleparticle characterization by microflow imaging (MFI). A summary of theresults is provided in Table 9. Provided in the left column is the testperformed and provided in the right column are the formulationssuggested based on the results of that test.

TABLE 9 Summary of results Test Suggested Candidates Visual AppearanceAll formulations (F1 to F21) Gel Permeation High F2 to F5, F10 to F21Performance Liquid Chromatography Reduced SDS PAGE F2 to F5, F8 to F21Non-reduced SDS PAGE F2 to F5, F8 to F21 Isoelectric Focusing Allformulations except F6, F7 and F21 A340-A620 All formulations (F1 toF21) Dynamic Light Scattering Lead: F5, F7 and F18-F21 Alternative: Allother formulations Differential Scanning Lead: F5, F7 and F18-F21Calorimetry Alternative: All other formulations Subvisible ParticleLead: All formulations except F16 Characterization by Microflow ImagingRecommended Buffer Ion & pH 25 mM histidine pH 6.5 RecommendedExcipients Sodium Chloride and Sucrose

Formulations were scored according to visual appearance. Samples at time0 were scored at 5° C. with no treatment, or with agitation (200 rpm onhorizontal plate shaker for 24 hours at ambient temperature) or withfreeze-thaw (3 cycles of −65° C. & below). Samples at 1 month werescored at 5° C. and at 40° C. with no treatment. All samples scored asclear colorless liquid or slightly opalescent, light yellow liquid.Samples were also scored according to particulate matter. All samplesscored as being free from visible particles, or having few particles.FIG. 3 shows the data from the visual appearance scoring.

Samples were also analyzed by gel permeation high performance liquidchromatography (GP HPLC). FIG. 4 shows the percentage of monomer,aggregates and fragments by GP HPLC. Samples at time 0 were analyzed at5° C. with no treatment, with agitation or with freeze-thaw. Samples at1 month were analyzed at 5° C. and at 40° C. with no treatment.Suggested candidates based on GP HPLC were F2 to F5 and F10 to F21.

Samples were also analyzed by reduced and non-reduced SDS PAGE. FIG. 5shows the relative percentage of IgG band area by reduced SDS PAGE andFIG. 6 shows the number of bands detected visually by non-reduced SDSPAGE. Samples at time 0 were analyzed at 5° C. with no treatment, orwith or with freeze-thaw. Samples at 1 month were analyzed at 5° C. andat 40° C. with no treatment. Suggested candidates based on reduced andnon-reduced SDS PAGE were F2 to F5 and F8 to F21.

Samples were analyzed by isoelectric focusing. FIG. 7 shows the numberof bands and pI range of samples stored at 5° C. with no treatment, withagitation or with freeze-thaw. FIG. 8 shows the number of bands and pIrange of samples stored at 40° C. The pI range was determined visuallyboth at start and finish against pI markers. Suggested candidates basedon isoelectric focusing were all formulations except F6, F7 and F21.

FIG. 9 shows the absorbance at 340 nm and at 620 nm for the differentformulations. Samples at time 0 were analyzed at 5° C. with notreatment, or with or with freeze-thaw. Samples at 1 month were analyzedat 5° C. and at 40° C. with no treatment. Suggested candidates based onabsorbance were all formulations (F1 to F21).

Dynamic light scattering was performed. The Rh(nm) by intensity, %polydispersity, and HMW species Rh (nm) were determined. Samples at time0 were analyzed at 5° C. with no treatment, or with or with freeze-thaw.Samples at 1 month were analyzed at 5° C. and at 40° C. with notreatment. The results are shown in FIG. 10. Suggested candidates basedon dynamic light scattering were F5, F7 and F18-F21, or in alternative,all other formulations.

Differential scanning calorimetry (DSC) was also performed. In FIG. 11,the melting temperatures at three different unfolding events are shownfor each formulation. Suggested candidates based on DSC were F5, F7 andF18-F21, or in alternative, all other formulations.

Sub-visible particle characterization by microflow imaging was alsoperformed. The results are shown in FIG. 12. Samples at time 0 and 1month at 5° C. with no treatment were analyzed. Particles ≧1 μm, ≧2 μm,≧5 μm, ≧10 μm, and ≧25 μm are reported. Suggested candidates based onsub-visible particle characterization were all formulations except F16.

Based on the information generated in this study, one of the selectedformulations for the anti-dengue antibodies described herein include ahistidine at pH 6.5. The selected excipients for use in this formulationwere sodium chloride, sucrose, and 0.02% polysorbate 80. Alternatively,the selected excipients for use in this formulation were sodiumchloride, arginine, and 0.02% polysorbate 80.

This selection was made based on overall assessment of the suggestedcandidates from the individual test methods shown in Table 9 anddescribed herein. Suggested candidates selected from each methodindicated that little or no marked changes had occurred to the productat +5° C. after one month storage and at the end of agitation and freezethaw studies compared to study start. In addition, product stored inthese suggested formulations also demonstrated the least changescompared to other formulations when stored at elevated temperature.

The overall data indicated that histidine was a more desirable bufferion compared to citrate, phosphate, and arginine. Also, pH 6.5 was amore desirable pH compared to pH 7.0.

The excipients were selected from formulations F14 and F15.

Formulation F14 (25 mM histidine pH 6.5, 75 mM NaCl, 100 mM sucrose) wasthe selected candidate for all test methods except for DSC and DLS.There were other candidates (F5, F12, F13) that had comparable data withF14. F14 was selected as one of the final candidates based on theoverall results including the data from sub-visible particle analysis.The data showed that F14 had one of the lowest particle counts (≧10 μmand ≧25 μm at T=0 and T=1M). In the DLS data for all formulations at+40° C. T=1M, only F19 and F21 did not display high molecular weightspecies (HMWS). However, as this was at an accelerated temperature andF14 did not display any HMWS for the +5° C. agitation, freeze thaw orT=0, and T=1M+5° C. samples, F14 was considered as a possible candidate.In the DSC data F14 was also considered as a candidate since there wasnot a large difference in the melting temperatures when compared to thealternative candidates.

Formulation F15 (25 mM histidine pH 6.5, 75 mM NaCl, 100 mM sucrose,0.02% polysorbate 80) was also selected for use in a formulation as thedata for all test methods were similar to F14. Polysorbate 80 (as asurfactant) is able to protect proteins from surface and stress-inducedaggregation. There were 2 HMWS detected at +40° C. by DLS in F15 ascompared to a single HMWS in F14. Although there was no confirmatorysub-visible particle data available for +40° C. at T=1M, thisformulation was selected given the known positive effects of includingpolysorbate 80 as an excipient.

Other formulations selected based on this study include, e.g.,formulation F12 (25 mM histidine pH 6.5, 75 mM NaCl, 75 mM arginine) andF13 (25 mM histidine pH 6.5, 75 mM NaCl, 75 mM arginine, 0.02%polysorbate 80).

Additional disclosures, including but not limited to, antibodysequences, compositions, methods, and examples, are disclosed inInternational Application Publication No. WO2015/122995. The content ofthe aforesaid publication is incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

All publications, patents, and accession numbers mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

EQUIVALENTS

While specific embodiments of the compositions and methods herein havebeen discussed, the above specification is illustrative and notrestrictive. Many variations of the invention will become apparent tothose skilled in the art upon review of this specification and theclaims below. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

What is claimed is:
 1. A formulation comprising 10 mg/mL to 50 mg/mL ofan anti-dengue antibody molecule, 10 mM to 50 mM histidine or sodiumcitrate, 50 mM to 150 mM sodium chloride, and 50 mM to 150 mM arginineor sucrose, wherein the formulation has a pH of 6 to 7, and wherein theantibody molecule comprises: (a) a heavy chain (HC) immunoglobulinvariable region segment comprising: an HC CDR1 comprising the sequenceDVYMS (SEQ ID NO: 3), an HC CDR2 comprising the sequenceRIDPENGDTKYDPKLQG (SEQ ID NO: 4), and an HC CDR3 comprising the sequenceGWEGFAY (SEQ ID NO: 5); and (b) a light chain (LC) immunoglobulinvariable region segment comprising: an LC CDR1 comprising the sequenceRASENVDKYGNSFMH (SEQ ID NO: 6), an LC CDR2 comprising the sequenceRASELQW (SEQ ID NO: 7), and an LC CDR3 comprising the sequence QRSNEVPWT(SEQ ID NO: 8).
 2. The formulation of claim 1, comprising 10 mg/mL to 40mg/mL of the antibody molecule.
 3. The formulation of claim 1,comprising 25 mg/mL of the antibody molecule.
 4. The formulation ofclaim 1, comprising 10 mM to 50 mM histidine.
 5. The formulation ofclaim 1, comprising 20 mM to 40 mM histidine.
 6. The formulation ofclaim 1, comprising 25 mM histidine.
 7. The formulation of claim 1,comprising 10 mM to 50 mM sodium citrate.
 8. The formulation of claim 1,comprising 10 mM to 30 mM sodium citrate.
 9. The formulation of claim 1,comprising 25 mM sodium citrate.
 10. The formulation of claim 1,comprising 75 mM to 150 mM sodium chloride.
 11. The formulation of claim1, comprising 75 mM sodium chloride.
 12. The formulation of claim 1,comprising 50 mM to 150 mM arginine.
 13. The formulation of claim 1,comprising 75 mM to 150 mM arginine.
 14. The formulation of claim 1,comprising 75 mM arginine.
 15. The formulation of claim 1, comprising 50mM to 150 mM sucrose.
 16. The formulation of claim 1, comprising 75 mMto 100 mM sucrose.
 17. The formulation of claim 1, comprising 100 mMsucrose.
 18. The formulation of claim 1, which has a pH of 6.5.
 19. Theformulation of claim 1, further comprising polysorbate
 80. 20. Theformulation of claim 1, further comprising 0.01% to 0.05% polysorbate80.
 21. The formulation of claim 1, further comprising 0.02% polysorbate80.
 22. The formulation of claim 1, wherein the antibody moleculecomprises the heavy chain variable region (VH) amino acid sequence ofSEQ ID NO:
 1. 23. The formulation of claim 1, wherein the antibodymolecule comprises the light chain variable region (VL) amino acidsequence of SEQ ID NO:
 2. 24. The formulation of claim 1, wherein theantibody molecule comprises the VH amino acid sequence of SEQ ID NO: 1and the VL amino acid sequence of SEQ ID NO:
 2. 25. The formulation ofclaim 1, comprising 10 mg/mL to 50 mg/mL of the antibody molecule, 10 mMto 50 mM histidine, 50 mM to 150 mM sodium chloride, and 50 mM to 150 mMsucrose, wherein the formulation has a pH of 6 to
 7. 26. The formulationof claim 1, comprising 25 mg/mL of the antibody molecule, 25 mMhistidine, 75 mM sodium chloride, and 100 mM sucrose, wherein theformulation has a pH of 6.5.
 27. The formulation of claim 1, comprising10 mg/mL to 50 mg/mL of the antibody molecule, 10 mM to 50 mM histidine,50 mM to 150 mM sodium chloride, 50 mM to 150 mM sucrose, 0.01% to 0.05%polysorbate 80, wherein the formulation has a pH of 6 to
 7. 28. Theformulation of claim 1, comprising 25 mg/mL of the antibody molecule, 25mM histidine, 75 mM sodium chloride, 100 mM sucrose, 0.02% polysorbate80, wherein the formulation has a pH of 6.5.
 29. The formulation ofclaim 1, comprising 10 mg/mL to 50 mg/mL of the antibody molecule, 10 mMto 50 mM histidine, 50 mM to 150 mM sodium chloride, and 50 mM to 150 mMarginine, wherein the formulation has a pH of 6 to
 7. 30. Theformulation of claim 1, comprising 25 mg/mL of the antibody molecule, 25mM histidine, 75 mM sodium chloride, and 75 mM arginine, wherein theformulation has a pH of 6.5.
 31. The formulation of claim 1, comprising10 mg/mL to 50 mg/mL of the antibody molecule, 10 mM to 50 mM histidine,50 mM to 150 mM sodium chloride, 50 mM to 150 mM arginine, 0.01% to0.05% polysorbate 80, wherein the formulation has a pH of 6 to
 7. 32.The formulation of claim 1, comprising 25 mg/mL of the antibodymolecule, 25 mM histidine, 75 mM sodium chloride, 75 mM arginine, 0.02%polysorbate 80, wherein the formulation has a pH of 6.5.
 33. Theformulation of claim 1, comprising 10 mg/mL to 50 mg/mL of the antibodymolecule, 10 mM to 50 mM sodium citrate, 50 mM to 150 mM sodiumchloride, and 50 mM to 150 mM sucrose, wherein the formulation has a pHof 6 to
 7. 34. The formulation of claim 1, comprising 25 mg/mL of theantibody molecule, 25 mM sodium citrate, 75 mM sodium chloride, and 75mM sucrose, wherein the formulation has a pH of 6.5.
 35. The formulationof claim 1, which is a liquid formulation.
 36. A container comprisingthe formulation of claim
 1. 37. A device comprising the formulation ofclaim
 1. 38. A kit comprising the formulation of claim 1, andinstructions for administration of the formulation to a subject.
 39. Amethod of treating or preventing dengue, the method comprisingadministering to a subject having dengue, or at risk of having dengue,an effective amount of the formulation of claim 1, thereby treating orpreventing dengue.
 40. A method of making a formulation comprising ananti-dengue antibody molecule, the method comprising combining theantibody molecule with (a) histidine or sodium citrate, (b) sodiumchloride, and (c) arginine or sucrose, wherein the antibody moleculecomprises: (a) a heavy chain (HC) immunoglobulin variable region segmentcomprising: an HC CDR1 comprising the sequence DVYMS (SEQ ID NO: 3), anHC CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4), and anHC CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5); and (b) a lightchain (LC) immunoglobulin variable region segment comprising: an LC CDR1comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6), an LC CDR2comprising the sequence RASELQW (SEQ ID NO: 7), and an LC CDR3comprising the sequence QRSNEVPWT (SEQ ID NO: 8), wherein theformulation comprises 10 mg/mL to 50 mg/mL of the antibody molecule, 10mM to 50 mM histidine or sodium citrate, 50 mM to 150 mM sodiumchloride, and 50 mM to 150 mM arginine or sucrose, and wherein theformulation has a pH of 6 to 7.